Method for producing transgenic plants with increased yield, comprising expressing of haemoglobin from Arabidopsis

ABSTRACT

The invention relates generally to a method for improving growth of plants, under normal and under stress conditions, more particularly under osmotic stress and/or temperature extremes, comprising modifying plant haemoglobin gene expression and/or by modifying plant haemoglobin protein levels in a plant. The invention furthermore relates to a method for increasing yield of a plant, comprising modifying plant gene expression and/or by modifying plant haemoglobin levels in a plant. The invention also relates to a nucleic acid encoding a haemoglobin conferring this altered growth and stress tolerance.

This application is the US national phase of international applicationPCT/EP2004/050405, filed 1 Apr. 2004, which designated the U.S. andclaims priority of EP 03075974.0, filed 1 Apr. 2003, the entire contentsof each of which are hereby incorporated by reference.

FIELD OF INVENTION

The present invention concerns a method for altering growthcharacteristics of plants, in particular a method for altering stresstolerance and yield in plants. More specifically, the present inventionconcerns altering tolerance in plants to various environmental stressesand to increasing seed yield by modifying plant haemoglobin geneexpression and/or by modifying plant haemoglobin protein levels.

BACKGROUND

Most varieties of crop plants available to agriculture today have beenobtained as a result of years of breeding activities focussed on theselection of higher yielding plants adapted to a particular environment.As a consequence, they often lack sufficient genetic variability toadapt to other environments whilst maintaining a high yield. Inaddition, during their life cycle, plants are exposed to variousenvironmental conditions which greatly influence development and which,when unfavourable, may limit the final yield. Climate and otherenvironmental conditions introduce variability into both totalproduction and in quality of the product obtained over differentseasons. Therefore it is a major aim in agriculture to develop varietieswith enhanced stability in a quantitative and qualitative sense.Stability in production in the quantitative sense would be beneficialfor planning and could avoid anomalies in production. In the qualitativesense, stability would contribute to improve post-harvest treatments andto industrial processing of agricultural products.

Yield is normally defined as the measurable produce of economic valuefrom a crop. This may be defined in terms of quantity and/or quality.Yield is directly dependent on several factors, for example, the numberand size of the organs, plant architecture (for example, the number ofbranches), seed production and more. Root development, nutrient uptakeand stress tolerance are also important factors influencing yield.

The ability to influence one or more of the abovementioned factors, andto thereby increase crop yield, would have many applications in areassuch as crop enhancement, plant breeding, production of ornamentalplants, arboriculture, horticulture, forestry, production of algae orplants (for example for use as bioreactors, for the production ofsubstances such as pharmaceuticals, antibodies, or vaccines, or for thebioconversion of organic waste or for use as fuel in the case ofhigh-yielding algae and plants).

The final yield of a plant is determined by several parameters amongstwhich growth is a major contributor. Often an increase in growthcorrelates with higher yield. Particularly relevant is the capacity of aplant to maintain growth and to continue its developmental programme inunfavourable conditions. Unfavourable conditions are those that limit aplant in achieving its potential maximum production. Given the plant'sinability of locomotion as a means of responding to environmentalstimuli, plants are exposed to a variety of stresses that limit theirperformance. Abiotic stress conditions, such as shortage or excess ofsolar energy, water and nutrients, extremes of hot and coldtemperatures, pollution (e.g. heavy-metal pollution) can all have amajor impact on plant growth and can significantly reduce plant yieldand growth.

The response of a plant to abiotic stresses, such as drought,temperature and osmotic stress, are intimately linked to each other (Zhuet al., Crit. Rev. Plant Sci. 16, 253-277, 1997). Many genes that areregulated by one type of stress are also responsive to the other two. Agene conferring tolerance to, for example, osmotic stress may thereforealso confer tolerance to cold and drought stresses. In addition, a plantcan be exposed to a multiplicity of stresses during its life cycle e.g.drought stress is often accompanied by high temperature stress. The mostcommon kind of stress plants receive from their surroundings istemperature stress. Each plant species has its own optimal temperaturefor growth, and its geographical distribution is determined to a majorextent by the temperature zone in which it can survive. Recently,concerns have been voiced about the potentially serious effects onagriculture of radical global temperature changes predicted to occur inthe near future. There is now an effort to search for practicalapproaches to improve adaptability of plants to non-optimal temperatureconditions. Molecular breeding methods have been applied to addressthese problems. For example, genetically engineered cold tolerance inplants has been achieved by overexpression of transcription factors suchas SCOF-1 or CBF1 (Kim et al., Plant J. 25, 247-259, 2001; Jaglo-Ottosenet al., Science 280, 104-106, 1998); increasing the content ofcompatible solutes, (Alia et al., Plant Cell Environm. 21, 232-239,1998); altering membrane lipids; and by reducing the effect of activeoxygen species. The ability to withstand high temperatures has beenobtained by engineering expression of heat shock proteins, increasingproduction of compatible solutes, and by altering membrane lipids.However to date there has been no scientific report describing theinvolvement of plant class-2 non-symbiotic haemoglobin genes inresponses to environmental stresses or of plant haemoglobin genes ingeneral in responses to temperature stresses.

Drought, salt stress and high or low temperature stress, are majorproblems in agriculture because these adverse environmental factorsprevent crop plants from maximally exploiting their genetic potential.These stresses influence virtually every aspect of plant physiology andmetabolism. Stress generally involves adaptive responses, such asmorphological changes in roots or other organs, but also developmentalchanges, e.g. inhibition of growth. In general the response of a plantcan be divided into three categories: maintenance of homeostasis, whichincludes ion homeostasis and osmotic homeostasis or osmotic adjustment;detoxification of harmful compounds, e.g. of reactive oxygen species orof damaged proteins that originated during the stress; and recovery ofgrowth, that is, relief from growth inhibition and the effects on celldivision and expansion imposed during the stress.

Progress has been made through genetic engineering in achieving stresstolerance by manipulating homeostasis, e.g. by increasing theconcentrations of osmolytes (Nuccio et al., Curr. Opin. Plant Biol. 2,128-34, 1999), by overexpressing Na+/H+ antiporters, (Apse and Blumwald,Curr. Opin. Biotechnol. 13, 146-50, 2002) or by overexpressing LEAproteins that may contribute to maintenance of membrane or proteinstability (Xu et al., Plant Physiol. 110, 249-257, 1996). Engineeringcomponents of the osmotic signalling pathway is also a promising routeto achieve osmotic stress tolerance. However, there is no report in theliterature establishing a crucial role of haemoglobin genes in improvingosmotic stress tolerance.

Haemoglobins are commonly found in a wide range of organisms (Vinogradovet al., Comp. Biochem. Physiol. 106, 1-26, 1993; Bolognesi et al., Prog.Biophys. Mol. Biol. 68, 29-68, 1997). With the possible exception ofbarley, all examined plant species have at least two haemoglobin genes.These genes have been reported to contain 3 conserved introns, a featureshared with animal haemoglobins (Arredondo-Peter et al., Plant Physiol.118, 1121-1125, 1998). Based on their structure, plant haemoglobins usedto be divided into two groups. The first is a group of symbiotichaemoglobins (leghaemoglobins), comprising haemoglobins that areabundantly present in infected cells of N₂-fixing nodules in leguminousplants but that can also be found in non-leguminous plants. The secondgroup comprises non-symbiotic haemoglobins, which are ancestral to thesymbiotic type of haemoglobins and which are more widespread in theplant kingdom.

In a more recent classification (Hunt et al., Plant Mol. Biol. 47,677-692, 2001), haemoglobins were grouped into class 1 or class 2,depending on their amino acid sequence. Haemoglobins that did not fitinto either class were assigned to a class 0 that was later renamed intoclass 3 (Wittenberg et al., J. Biol. Chem. 277: 871-874, 2002). Becausethe different classes are delineated based on primary amino acidsequences, symbiotic haemoglobins and non-symbiotic haemoglobins may befound in both class 1 or 2. Class 3 comprises the truncatedhaemoglobins. Members of these three classes not only differ in aminoacid sequence, but also in biochemical properties. Truncatedhaemoglobins are small proteins carrying a haeme group that is able tobind oxygen. Class 1 and class 2 haemoglobins can be discriminated fromeach other in the conservation of certain amino acids in the sequence(see Hunt et al., 2001 for a detailed description of classes 1 and 2).Class 2 haemoglobins have conserved proline residues at positions B3 andG3, the absence of which may cause a different orientation in the B andG helices in class 1 haemoglobins. Additional substitutions and changesin charge at certain positions in the sequence cause furthermodifications in the packing of these helices.

The symbiotic haemoglobins are predominantly found in nodules ofleguminous and in non-leguminous plants living symbiotically withbacteria. In plants, symbiotic haemoglobins are known to play a role inoxygen transport, thereby stimulating nitrogen fixation by providingoxygen to the nodules. This is made possible by the high affinity foroxygen that the leghaemoglobins have, combined with a fast dissociationconstant for oxygen (Appleby, Sci. Prog. 76, 365-398, 1992).Leghaemoglobins belong to a multigene family and are usuallyposttranslationally modified. The bacterial haemoglobin fromVitreoscilla sp. resembles the leghaemoglobins in its binding propertiesfor oxygen and because of this property the protein has been used topromote growth in plants and micro-organisms (U.S. Pat. No. 5,049,493;U.S. Pat. No. 5,959,187). Vitreoscilla haemoglobin has a K_(D) of 6000nM, whereas the Arabidopsis class-2 haemoglobin has a K_(D) of 130 nM,which is more than 45 times lower. This high K_(D) makes Vitreoscillahaemoglobin well suited for stimulating oxygen transport andconsequently plant growth. Therefore, Vitreoscilla haemoglobin is quitedistinct from plant non-symbiotic haemoglobins (Bülow et al., TrendsBiotechnol. 17, 21-24, 1999). The use of Vitreoscilla haemoglobin may becompared to the use of bovine haemoglobin for promoting oxygen transferin plant cell culture media and for plant regeneration (Azhakanandam etal., Enzyme Microb. Technol. 21, 572-577, 1997).

The non-symbiotic haemoglobins on the other hand differ from theleghaemoglobins in their primary protein structure (Arredondo-Peter etal., 1998). In addition, non-symbiotic plant haemoglobins have a veryhigh affinity for oxygen, with a moderate association constant and avery low dissociation constant (about 40 times lower than thedissociation constant for oxygen of symbiotic haemoglobins(Arredondo-Peter et al., 1998; Bülow et al., 1999; Watts et al., Proc.Natl. Acad. Sci USA 98, 10119-10124)). Consequently oxygen is stablybound and a role in oxygen sensing or oxygen transport is not likely(Arredondo-Peter et al., 1998). However little is known about thefunctions in planta of the non-symbiotic haemoglobins. Their biochemicalproperties seem to exclude a role in oxygen diffusion, though a role asoxygenase may be possible (Hill, Can. J. Bot. 76, 707-712, 1998). Thebinding of oxygen causes a conformational change that may affectassociated ligand molecules, thereby triggering certain physiologicalresponses (Goodman and Hargrove, J. Biol. Chem. 276, 6834-6839, 2001).

Class 1 haemoglobins are induced by hypoxia, increasing sucroseconcentrations (Trevaskis et al., Proc. Natl. Acad. Sci. USA 94,12230-12234, 1997) or by nitrates (Wang et al., Plant Cell 12,1491-1510, 2000). They are also expressed in germinating seeds and inroots of mature plants (Hunt et al., 2001) and in differentiating cells(Ross et al., Protoplasma 218, 125-133, 2001). Class 1 non-symbiotichaemoglobins are induced upon hypoxic stress (Hunt et al., 2001).Arabidopsis haemoglobin 1 enhances survival under hypoxic stress andpromotes early shoot and root growth in Arabidopsis thaliana (Hunt etal., Proc. Natl. Acad. Sci. USA 99, 17197-17202, 2002). The use ofclass-1 haemoglobin molecules for altering plant growth characteristicswas mainly focused towards manipulating oxygen levels in the plant.Tarczynski and Shen (U.S. Pat. No. 6,372,961) propose the use of maizehaemoglobin to modify the oxygen concentrations in a plant cell and tostimulate seed germination and seedling growth of plants. Similarly,overexpression of haemoglobin from barley was shown to increase the ATPcontent in maize cells and to maintain the energy status under hypoxicstress (Guy et al., WO 00/00597).

The expression pattern of class-2 haemoglobins is different from that ofclass 1 haemoglobins in that they are expressed during embryogenesis andseed maturation, around openings (e.g. in mesophyl cells of stomata,around the top of the style, around the pore of the nectaries) or atbranch points (e.g. to the bolt system, around emerging lateral roots,at the junction of anther and filament) (Hunt et al., 2001). Members ofthe class-2 haemoglobins are also responsive to cytokinin (Hunt et al.,2001). Harper et al. (WO 02/16655) have shown that haemoglobin 2 isinduced in Arabidopsis upon cold, osmotic and saline stress, togetherwith over 400 other genes. However, this class-2 haemoglobin has notbeen linked to increased stress tolerance. To date, only a few class-2haemoglobin sequences have been described, among which is the GLB2 fromArabidopsis thaliana and two ESTs from Beta vulgaris that were isolatedfrom stressed seedlings (GenBank acc no BE590299) and from a leaf cDNAlibrary (GenBank acc no BQ586966).

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, the inventors have now demonstrated that plant class-2non-symbiotic haemoglobins are useful for increasing the yield of aplant and for increasing abiotic stress. Therefore and according to afirst embodiment of the present invention, there is provided a methodfor altering plant characteristics selected from one or more ofincreased yield, increased biomass, or altered cell division of a plant,comprising increasing expression in a plant of a nucleic acid sequenceencoding plant class-2 non-symbiotic haemoglobin. Preferably, theincrease in yield comprises increased seed yield.

The term “increased yield” or “increased biomass” encompasses anincrease in biomass in one or more parts of a plant relative to thebiomass of corresponding wild-type plants. The term also encompasses anincrease in seed yield, which includes an increase in the biomass of theseed (which may be represented as weight of individual seeds or as totalseed weight) and/or an increase in the number of (filled) seeds and/orin the size of the seeds and/or an increase in seed volume, eachrelative to corresponding wild-type plants. An increase in seed sizeand/or volume may also influence the composition of seeds. An increasein seed yield may be due to an increase in the number and/or size offlowers. An increase in yield may also increase the harvest index, whichis expressed as a ratio of the total biomass over the yield ofharvestable parts, such as seeds. The harvested part of a plant maydiffer from crop to crop, for example it may be seed (in the case ofrice, sorghum or corn grown for seed); it may be above-ground biomass(in the case of corn, used as silage, or sugarcane), root (e.g. sugarbeet), fruit (e.g. tomato), cotton fibres, or any other part of theplant which is of economic value. For example, the methods of thepresent invention may be used to increase the seed yield of rice andcorn or to increase yield of silage corn in terms of overall aboveground biomass and energy content. An increase in yield also encompassesa better performance of the plant under non-stress conditions or understress conditions compared to wild-type plants. Stress conditionsinclude any type of environmental stress and biotic and abioticstresses.

The term “modified cell division” encompasses an increase or decrease incell division or an abnormal cell division/cytokinesis, altered plane ofdivision, altered cell polarity, altered cell differentiation. Modifiedcell division may also give rise to altered cell size and cell number.Modified cell division in a plant may furthermore result in modifiedgrowth of that plant.

The term “modified plant growth” as used herein encompasses, but is notlimited to, a faster rate of growth in one or more parts of a plant(including seeds), at one or more stages in the life cycle of a plant(including germination), each relative to corresponding wild-typeplants. Increased growth rate during the early stages in the life cycleof a plant may give rise to enhanced vigour, compared to correspondingwild-type plants. According to a preferred feature of the presentinvention, the faster growth rate takes place during substantially themajority of the plant's life cycle. An increase in growth rate may alsoalter the harvest time of a plant allowing plants to be harvested soonerthan would otherwise be possible. If the growth rate is sufficientlyincreased, it may even give rise to the possibility of sowing furtherseeds of the same plant species (for example sowing and harvesting ofrice plants followed by sowing and harvesting of further rice plants allwithin one conventional growing period) or of different plants species(for example the sowing and harvesting of rice plants followed by, forexample, the sowing and optional harvesting of soy bean, potatoes or anyother suitable plant), thereby increasing the annual biomass productionper acre (due to an increase in the number of times (say in a year) thatany particular plant may be grown and harvested).

According to another embodiment of this invention, there is provided amethod for altering architecture of a plant, comprising increasingexpression in a plant of a nucleic acid sequence encoding planthaemoglobin, preferably a non-symbiotic haemoglobin, more preferably aclass-2 non-symbiotic haemoglobin.

“Altered architecture” may be due to a change in cell division. The term“architecture” as used herein encompasses the appearance or morphologyof a plant, including any one or more structural features or acombination of structural features. Such structural features include theshape, size, number, position, texture, arrangement, and pattern of anycell, tissue or organ or groups of cells, tissues or organs of a plant,including the root, leaf, shoot stem, petiole, trichome, flower,inflorescence (for monocotyledonous and dicotyledonous plants),panicles, petal, stigma, style, stamen, pollen, ovule, seed, embryo,endosperm, seed coat, aleurone, fibre, cambium, wood, heartwood,parenchyma, aerenchyma, sieve element, phloem or vascular tissue,amongst others. Modified architecture therefore includes all aspects ofmodified growth of the plant. Sometimes plants modify their architecturein response to certain conditions such as stress and pathogens (e.g.nematodes). Therefore, within the scope of the term “architecture” isincluded modified architecture under stress conditions, whether bioticor abiotic stress conditions.

According to a further embodiment of the present invention, a method isprovided for increasing stress tolerance of a plant, preferably abioticstress tolerance, comprising increasing expression in a plant of anucleic acid sequence encoding plant class-2 non-symbiotic haemoglobin.

“Increased stress tolerance” as used herein comprises, for any givenstress, increasing tolerance in plants to that particular stress,whether those plants already have some degree of tolerance to theparticular stress or whether that plant is being provided with toleranceto that stress anew. The altered stress tolerance is preferably alteredtolerance to various abiotic stresses. Abiotic stresses are caused byelements present in the environment, which may include, but are notlimited to: osmotic stress, drought, salt, dehydration, freezing, heat,cold, water logging, wounding, mechanical stress, oxidative stress,ozone, high light, heavy metals, nutrient deprivation, toxic chemicalsand combinations of the same. Some of these stresses can also occur as aconsequence of infection by pathogens (such as viruses, bacteria, fungi,insects or nematodes). According to a preferred feature of the presentinvention, increased abiotic stress tolerance is increased tolerance toosmotic stress.

In the case where abiotic stress is high temperature stress, any planthaemoglobin can be used for increasing stress tolerance. Therefore,there is provided a method for increasing high temperature stresstolerance of a plant, comprising increasing expression in a plant of anucleic acid sequence encoding plant haemoglobin, preferably anon-symbiotic haemoglobin, more preferably a class-2 non-symbiotichaemoglobin.

The term “high temperature stress” refers to a stress condition causedby temperatures that are above the optimal growth temperature for agiven plant. The term “increased tolerance” includes the capacity of aplant to endure any given stress to a greater degree than correspondingwild type plants. This may be manifested by, say, improved growth orsurvivability of the plant relative to corresponding wild type plants.The term also includes faster resumption of growth and/or developmentfollowing a period of stress. It may also be that in certainapplications it would be advantageous to decrease the tolerance in aplant to a particular type of stress. Altered stress tolerance thereforealso includes decreasing tolerance in a plant to any given stress, i.e.making the plant more susceptible to any given stress. This may beadvantageous for, for example, the production of certain metabolites.

Performance of the methods according to the present inventionadvantageously results in plants having altered growth characteristics,comprising increased yield and/or modified cell division and/or alteredarchitecture and/or increased stress tolerance, in particular increasedosmotic stress tolerance and/or high temperature stress tolerance.

According to preferred feature of the present invention, the haemoglobinuseful in the methods according to the invention is a plant haemoglobin,preferably a non-symbiotic haemoglobin from a dicotyledonous plant.Further preferably the non-symbiotic haemoglobin which is derived from adicotyledonous plant is a non-symbiotic class-2 haemoglobin. Theclassification of haemoglobins into class 1 or 2 is according to Hunt etal. (2001).

Advantageously, the methods according to the present invention foraltering architecture or for increasing yield, biomass and/or celldivision may be practised with a class-2 non-symbiotic haemoglobin froma dicotyledonous plant Preferably, the class-2 non-symbiotic haemoglobinoriginates from a member of the Brassicaceae, such as Arabidopsisthaliana, most preferably, the class-2 non-symbiotic haemoglobin isencoded by a sequence essentially similar to any one of SEQ ID NO 1, 3or 5, or is a protein essentially similar to any one of SEQ ID NO 2, 4,6, or is a homologous plant class-2 non-symbiotic haemoglobin with atleast 65% sequence identity to SEQ ID NO 2.

Advantageously, where the plant characteristic to be altered is stresstolerance, the haemoglobin may be any class-2 non-symbiotic haemoglobinfrom a dicotyledonous plant, but preferably the class-2 non-symbiotichaemoglobin is isolated from the Brassicaceae, more preferably from Betavulgaris, most preferably, the class-2 non-symbiotic class-2 haemoglobinis encoded by a nucleic acid sequence essentially similar to SEQ ID NO1, or is a protein essentially similar to SEQ ID NO 2, or is ahomologous plant class-2 non-symbiotic haemoglobin with at least 65%sequence identity to SEQ ID NO 2.

The sequences encoding the complete non-symbiotic haemoglobin from Betavulgaris were hitherto unknown. Therefore according to another aspect ofthe present invention, there is provided an isolated nucleic acidsequence encoding a plant class-2 non-symbiotic haemoglobin selectedfrom:

-   -   (i) a nucleic acid sequence comprising a sequence according to        SEQ ID NO 1 or the complement thereof;    -   (ii) a nucleic acid sequence encoding a protein with an amino        acid sequence which is at least, in increasing order of        preference, 79%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%        identical to the amino acid sequence as given in SEQ ID NO 2;    -   (iii) a nucleic acid encoding a protein comprising the amino        acid sequence as given in SEQ ID NO 2;    -   (iv) a nucleic acid according to any of (i) to (iii) which is        degenerate as a result of the genetic code;    -   (v) a nucleic acid which is a splice variant of a nucleic acid        according to any of (i) to (iv);    -   (vi) a nucleic acid which is divergent due to differences        between alleles encoding a protein as given in SEQ ID NO 2, or        as defined in (i) to (v);    -   (vii) a nucleic acid encoding an immunologically active and/or        functional fragment of a protein encoded by a DNA sequence        according to any of (i) to (vi); and    -   (viii) a nucleic acid sequence which hybridises, preferably        under stringent conditions, to sequences defined in (i) to        (vii),        with the proviso that none of (i) to (viii) include the sequence        as given in GenBank acc no BE590299 or BQ586966.

The nucleic acid sequence as set forth in SEQ ID NO 1 (also referred toas clone BvXero2), encodes a class-2 haemoglobin from Beta vulgaris.Advantageously, the methods according to the present invention may alsobe practised using nucleic acids (i) to (viii). These nucleic acidsencompass nucleic acids encoding homologues, derivatives and functionalfragments of the protein encoded by the sequence depicted in SEQ IDNO 1. The term also includes at least a part of SEQ ID NO 1; acomplement of the sequence presented by SEQ ID NO 1; RNA, DNA, cDNA orgenomic DNA corresponding to the sequence of SEQ ID NO 1; a variant ofSEQ ID NO 1 due to the degeneracy of the genetic code; a family memberof the gene or protein; an allelic variant of SEQ ID NO 1; differentsplice variants and variants of SEQ ID NO 1 that are interrupted by oneor more intervening sequences.

The terms “gene(s)”, “polynucleotide(s)”, “nucleic acid sequence(s)”,“nucleotide sequence(s)”, “DNA sequence(s)” or “nucleic acidmolecule(s)”, as used herein refers to nucleotides, eitherribonucleotides or deoxyribonucleotides or a combination of both, in apolymeric form of any length. These terms furthermore includedouble-stranded and single-stranded DNA and RNA. These terms alsoinclude known nucleotide modifications such as methylation, cyclisationand ‘caps’ and substitution of one or more of the naturally occurringnucleotides with an analogue such as inosine. The terms also encompasspeptide nucleic acids (PNAs), a DNA analogue in which the backbone is apseudopeptide consisting of N-(2-aminoethyl)-glycine units rather than asugar. PNAs mimic the behaviour of DNA and bind complementary nucleicacid strands. The neutral backbone of PNA results in stronger bindingand greater specificity than normally achieved. In addition, the uniquechemical, physical and biological properties of PNA have been exploitedto produce powerful biomolecular tools, antisense and antigene agents,molecular probes and biosensors. With “recombinant” DNA molecule ismeant a hybrid DNA produced by joining pieces of DNA from differentsources. With “heterologous” nucleotide sequence is intended a sequencethat is not naturally occurring with the promoter sequence. While thisnucleotide sequence is heterologous to the promoter sequence, it may behomologous, or native, or heterologous, or foreign, to the plant host.

A “coding sequence” or “open reading frame” or “ORF” is defined as anucleotide sequence that can be transcribed into mRNA and/or translatedinto a polypeptide when placed under the control of appropriateregulatory sequences, i.e. when the coding sequence or ORF is present inan expressible format. The coding sequence or ORF is bound by a 5′translation start codon and a 3′ translation stop codon. A codingsequence or ORF may include RNA, mRNA, cDNA, recombinant nucleotidesequences, synthetically manufactured nucleotide sequences or genomicDNA. The coding sequence or ORF may be interrupted by interveningnucleic add sequences.

Genes and coding sequences encoding substantially the same protein butisolated from different sources may consist of substantially divergentnucleic acid sequences. Reciprocally, substantially divergent nucleicadd sequences may be designed to effect expression of essentially thesame protein. These nucleic acid sequences are the result of e.g. theexistence of different alleles of a given gene, or of the degeneracy ofthe genetic code or of differences in codon usage. Differences inpreferred codon usage are illustrated in http://www.kazusa.or.jp/codon.Allelic variants are further defined as to comprise single nucleotidepolymorphisms (SNPs) as well as small insertion/deletion polymorphisms(INDELs; the size of INDELs is usually less than 100 bp). SNPs andINDELs form the largest set of sequence variants in naturally occurringpolymorphic strains of most organisms. Additionally or alternatively, inparticular conventional breeding programs, such as for example markerassisted breeding, it is sometimes practical to introduce allelicvariation in the plants by mutagenic treatment of a plant. One suitablemutagenic method is EMS mutagenesis. Identification of allelic variantsthen takes place by, for example, PCR. This is followed by a selectionstep for selection of superior allelic variants of the sequence inquestion and which give rise to altered growth characteristics.Selection is typically carried out by monitoring growth performance ofplants containing different allelic variants of the sequence in question(for example, SEQ ID NO 1). Monitoring growth performance may be done ina greenhouse or in the field. Further optional steps include crossingplants, in which the superior allelic variant was identified, withanother plant. This could be used, for example, to make a combination ofinteresting phenotypic features.

According to another aspect of the present invention, advantage may betaken of the nucleotide sequence capable of altering expression of anucleic acid encoding haemoglobin in breeding programs. For example, insuch a program, a DNA marker is identified which may be geneticallylinked to a gene capable of altering the activity of haemoglobin in aplant (which gene may be a gene encoding a haemoglobin or another genecapable of influencing the activity of a haemoglobin). This DNA markeris then used in breeding programs to select plants having altered growthcharacteristics. Many techniques are nowadays available to identify SNPsand/or INDELs.

“Hybridisation” is a process wherein substantially homologouscomplementary nucleotide sequences anneal to each other. Thehybridisation process may occur entirely in solution, i.e. bothcomplementary nucleic acids are in solution. Tools in molecular biologyrelying on such a process include the polymerase chain reaction (PCR;and all methods based thereon), subtractive hybridisation, random primerextension, nuclease S1 mapping, primer extension, reverse transcription,cDNA synthesis, differential display of RNAs, and DNA sequencedetermination. The hybridisation process may also occur with one of thecomplementary nucleic acids immobilised to a matrix such as magneticbeads, Sepharose beads or any other resin. Tools in molecular biologyrelying on such a process include the isolation of poly (A+) mRNA. Thehybridisation process may furthermore occur with one of thecomplementary nucleic acids immobilised to a solid support such as anitrocellulose or nylon membrane or immobilised by e.g. photolithographyto e.g. a siliceous glass support (the latter known as nucleic acidarrays or microarrays or as nucleic acid chips). Tools in molecularbiology relying on such a process include RNA and DNA gel blot analysis,colony hybridisation, plaque hybridisation, in situ hybridisation andmicroarray hybridisation. In order to allow hybridisation to occur, thenucleic acid molecules are generally thermally or chemically denaturedto melt a double strand into two single strands and/or to removehairpins or other secondary structures from single stranded nucleicacids. The stringency of hybridisation is influenced by conditions suchas temperature, salt concentration and hybridisation buffer composition.High stringency conditions for hybridisation include high temperatureand/or low salt concentration (salts include NaCl and Na₃-citrate)and/or the inclusion of formamide in the hybridisation buffer and/orlowering the concentration of compounds such as Sodium Dodecyl Sulphate(SDS) in the hybridisation buffer and/or exclusion of compounds such asdextran sulphate or polyethylene glycol (promoting molecular crowding)from the hybridisation buffer. Conventional hybridisation conditions aredescribed e.g. (Sambrook et al. 2001) but the skilled craftsman willappreciate that numerous different hybridisation conditions may bedesigned in function of the known or expected homology and/or length ofthe nucleic acid sequence. Sufficiently low stringency hybridisationconditions are particularly preferred to isolate nucleic addsheterologous to the DNA sequences of the invention defined supra.Elements contributing to the heterology include allelism, degenerationof the genetic code and differences in preferred codon usage asdiscussed supra.

The invention also relates to DNA sequences hybridising under stringentconditions to the DNA sequences according to the invention with theproviso that the hybridising DNA sequence as given in GenBank acc noBE590299 or BQ586966 is excluded.

DNA sequences may also be interrupted by intervening sequences. With“intervening sequences” is meant any nucleic acid sequence whichdisrupts a coding sequence comprising the DNA sequence according to theinvention or which disrupts the expressible format of a DNA sequencecomprising the DNA sequence according to the invention. Removal of theintervening sequence restores the coding sequence or the expressibleformat. Examples of intervening sequences include introns, mobilisableDNA sequences such as transposons and DNA tags such as a T-DNA. With“mobilisable DNA sequence” is meant any DNA sequence that may bemobilised as a result of a recombination event.

The term “fragment of a sequence” means a truncated version of thesequence in question. The truncated sequence (nucleic acid or proteinsequence) may vary widely in length while the maximum size is notcritical. Typically, the truncated amino acid will range from about 5 toabout 60 amino acids in length. In case of a “functional fragment”, theminimum size is a sequence of sufficient size to provide this sequencewith at least a comparable function and/or activity to the originalsequence which was truncated, The functionality of haemoglobin may bedetermined for example by spectroscopy or by kinetic analysis of O₂binding (Arredondo-Peter et al., 1997).

“Immunologically active” refers to molecules or specific fragmentsthereof, such as specific epitopes or haptens, that are recognised by(i.e. that bind to) antibodies. Specific epitopes may be determinedusing, for example, peptide-scanning techniques as described in Geysenet al., Chem Biol., 3 (8), 679-88, 1996. Functional fragments may alsoinclude those comprising an epitope which is specific for the proteinsaccording to the invention.

The present invention also provides an isolated plant class-2non-symbiotic haemoglobin protein comprising one of the polypeptidesselected from:

-   -   a a polypeptide as represented by SEQ ID NO 2;    -   b a polypeptide with an amino acid sequence which is at least,        in increasing order of preference, 79%, 80%, 85%, 90%, 95% 96%,        97%, 98% and 99% identical to the amino acid sequence as given        in SEQ ID NO: 2;    -   c a polypeptide encoded by a nucleic acid sequence as defined        above;    -   d a homologue, derivative, immunologically active and/or        functional fragment of a protein as defined in any of (i) to        (iii).

Advantageously, proteins according to any of a to d above may be used inthe methods of the present invention. Proteins essentially similar tothe protein according to the invention comprise at least a part of SEQID NO 2, functional fragments, homologues, derivatives, substitutionalvariants, deletional variants and insertional variants of SEQ ID NO 2,as well as the protein presented in SEQ ID NO 2 itself.

The terms “protein(s)”, “peptide(s)”, “polypeptide(s)” or“oligopeptide(s)”, when used herein refer to amino acids in a polymericform of any length. These terms also include known amino acidmodifications such as disulphide bond formation, cysteinylation,oxidation, glutathionylation, methylation, acetylation, farnesylation,biotinylation, stearoylation, formylation, lipoic acid addition,phosphorylation, sulphation, ubiquitination, myristoylation,palmitoylation, geranylgeranylation, cyclization (e.g. pyroglutamic acidformation), oxidation, deamidation, dehydration, glycosylation (e.g.pentoses, hexosamines, N-acetylhexosamines, deoxyhexoses, hexoses,sialic acid etc.), acylation and radiolabelling (e.g. with ¹²⁵I, ¹³¹I,³⁵S, ¹⁴C, ³²P, ³³P, ³H) as well as non-naturally occurring amino acidresidues, L-amino acid residues and D-amino acid residues.

“Homologues” of a protein of the invention are those peptides,oligopeptides, polypeptides, proteins and enzymes which contain aminoacid substitutions, deletions and/or additions relative to a proteinwith respect to which they are a homologue, without altering one or moreof its functional properties, in particular without reducing theactivity of the resulting product. For example, a homologue of a proteinwill consist of a bioactive amino acid sequence variant of the protein.To produce such homologues, amino acids present in the protein may bereplaced with other amino acids having similar properties, for examplehydrophobicity, hydrophilicity, hydrophobic moment, antigenicity,propensity to form or break alpha-helical structures or beta-sheetstructures, and so on.

Substitutional variants of a protein of the invention are those in whichat least one residue in the protein amino acid sequence has been removedand a different residue inserted in its place. Amino acid substitutionsare typically of single residues, but may be clustered depending uponfunctional constraints placed upon the polypeptide; insertions willusually be of the order of about 1-10 amino acid residues and deletionswill range from about 1-20 residues. Preferably, amino acidsubstitutions will comprise conservative amino acid substitutions.

Insertional amino acid sequence variants of a protein of the inventionare those in which one or more amino acid residues are introduced into apredetermined site in a protein. Insertions may comprise amino-terminaland/or carboxy-terminal fusions as well as intra-sequence insertions ofsingle or multiple amino acids. Generally, insertions within the aminoacid sequence will be smaller than amino or carboxyl terminal fusions,for example of the order of about 1 to 10 residues. Examples of amino-or carboxy-terminal fusion proteins or peptides include the bindingdomain or activation domain of a transcriptional activator as used inthe yeast two-hybrid system, phage coat proteins, (histidine)₆-tag,glutathione S-transferase, protein A, maltose-binding protein,dihydrofolate reductase, Tag•100 epitope (EETARFQPGYRS (SEQ ID NO:13)),c-myc epitope (EQKLISEEDL (SEQ ID NO:14)), FLAG-epitope (DYKDDDK (SEQ IDNO:15)), lacZ, CMP (calmodulin-binding peptide), HA epitope (YPYDVPDYA(SEQ ID NO:16)), protein C epitope (EDQVDPRLIDGK (SEQ ID NO:17)) and VSVepitope (YTDIEMNRLGK (SEQ ID NO:18)).

Deletional variants of a protein of the invention are characterised bythe removal of one or more amino acids from the amino acid sequence ofthe protein.

Amino acid variants of a protein of the invention may readily be madeusing peptide synthetic techniques well known in the art, such as solidphase peptide synthesis and the like, or by recombinant DNAmanipulations. The manipulation of DNA sequences to produce variantproteins, which are manifested as substitutional, insertional ordeletional variants are also well known in the art. For example,techniques for making substitution mutations at predetermined sites inDNA having a known sequence are well known to those skilled in the art,such as by M13 mutagenesis, T7-Gen in vitro mutagenesis kit (USB,Cleveland, Ohio), QuickChange Site Directed mutagenesis kit (Stratagene,San Diego, Calif.), PCR-mediated site-directed mutagenesis or othersite-directed mutagenesis protocols. Another alternative to manipulateDNA sequences to produce variant proteins, which are manifested assubstitutional, insertional or deletional variants, comprises targetedin vivo gene modification which may be achieved by chimeric RNA/DNAoligonucleotides as described by e.g. (Palmgren, Trends Genetics 13 (9),348, 1997; Yoon et al., Proc. Natl. Acad. Sci U.S.A., 93 (5), 2071-2076,1996).

“Derivatives” of a protein of the invention are those peptides,oligopeptides, polypeptides, proteins and enzymes which comprise atleast about five contiguous amino acid residues of the polypeptide butwhich retain the biological activity of the protein. A “derivative” mayfurther comprise additional naturally-occurring, altered glycosylated,acylated or non-naturally occurring amino acid residues compared to theamino acid sequence of a naturally-occurring form of the polypeptide.Alternatively or in addition, a derivative may comprise one or morenon-amino add substituents compared to the amino acid sequence of anaturally occurring form of the polypeptide, for example a reportermolecule or other ligand, covalently or non-covalently bound to theamino acid sequence such as, for example, a reporter molecule which isbound thereto to facilitate its detection.

Methods for the search and identification of homologues of thehaemoglobin are known to a person skilled in the art. Methods for thealignment of sequences for comparison are also well known in the art,such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. The BLASTalgorithm calculates percent sequence identity and performs astatistical analysis of the similarity between the two sequences. Thesoftware for performing BLAST analysis is publicly available through theNational Centre for Biotechnology information. GAP uses the algorithm ofNeedleman and Wunsch (J. Mol. Biol. 48: 443-453, 1970) to find thealignment of two complete sequences that maximises the number of matchesand minimises the number of gaps. When using the Needleman-Wunschalgorithm for determining the percentage identity between two proteinsequences, the full length sequences are preferably used in combinationwith the BLOSUM62 matrix, a gap opening penalty of 10 or 11 and a gapextension penalty of 0.5 or 1.

The cDNA insert of the plasmid pYPGEXERO2 present in clone BvXero2contains an 860 bp cDNA (SEQ ID NO 1, named BvXERO2) with an openreading frame of 456 base pairs encoding a polypeptide of 152 aminoacids (SEQ ID NO 2) with a predicted molecular weight of 16.72 kD. Thispolypeptide, named Xero2, comprises the amino acid residues that areconserved for all plant haemoglobins. These include the cd1phenylalanine, C2 proline and F8 proximal histidine residues needed forhaeme binding and the E7 distal histidine which is involved in ligandbinding in many classes of haemoglobin. This nomenclature is inaccordance with the three dimensional structure naming system used foranimal haemoglobins (Dickerson and Geis, Hemoglobin: Structure,function, Evolution and Pathology, Benjamin-Cummings, Menlo Park, USA),wherein helices are designated by an upper-case letter and interhelicaldomains are designated by two lower-case letters. The number refers tothe position along the domain starting at the N-terminal end. BvXero2falls in the group GLB2, described for non-symbiotic plant haemoglobins.This group contains the conserved residues His (E7), His (F8) and Phe(cd11) (Hunt et al., 2001 and Trevaskis et al., 1997). Expressionanalysis of the ARAth GLB2 promoter show that expression is localised inroots, leaves and inflorescence and may be induced in young plants bycytokinin treatment, but is not induced by abscisic acid,2,4-dichlorophenoxyacetic acid, giberellic acid, benzo (1,2,3)thiadiazole-7-carbothioic acid S-methyl ester or methyl jasmonate (Huntet at, 2001).

This polypeptide may be produced by introducing into a host cell anisolated nucleic acid molecule or a nucleic acid construct according tothe invention as described below, culturing the host cell underconditions allowing expression of the polypeptide and recovering theproduced polypeptide from the culture.

Further advantageously, the methods of the present invention areapplicable to organisms other than plants, for example the invention isapplicable to yeast and bacteria. The ability to provide yeast withtolerance to various stresses may have many economic advantages relevantto the baking industry, the brewing industry and others. Tolerance toheat and osmotic stresses are of particular economic advantage.Similarly, the ability to provide bacteria with tolerance to variousstresses may also be advantageous. For example, bacteria or yeasts withenhanced tolerance to osmotic and heat stress may be particularly suitedfor large-scale fermentation processes, as they allow the use of moreconcentrated nutritive media and are better adapted against the heatproduced by the metabolic processes in such fermentation. The presentinvention thus also provides a host cell comprising a nucleic acidsequence or nucleic acid construct as described above, wherein the hostcell is a bacterial, yeast, fungal, plant or animal cell. The isolatedpolypeptide may also be produced by chemical synthesis.

Advantageously, altering expression of a nucleic acid sequence encodinga haemoglobin and/or altering of activity of the haemoglobin itself maybe effected by chemical means, i.e. by exogenous application of one ormore compounds or elements capable of altering activity of thehaemoglobin and/or capable of altering expression of a haemoglobin gene(which may be either an endogenous gene or a transgene introduced into aplant). The exogenous application may comprise contacting oradministering cells, tissues, organs or organisms with the gene productor a homologue, derivative or active fragment thereof and/or toantibodies to the gene product. Such antibodies may comprise“plantibodies”, single chain antibodies, IgG antibodies and heavy chaincamel antibodies, as well as fragments thereof. Altering expression of anucleic acid sequence encoding a haemoglobin and/or altering of activityof the haemoglobin itself may also be effected as a result of decreasedlevels of factors that directly or indirectly activate or inactivate ahaemoglobin. Additionally or alternatively, contacting or administeringcells, tissues, organs or organisms with an interacting protein or to aninhibitor or activator of the gene product provides another exogenousmeans for altering expression of a nucleic acid sequence encoding ahaemoglobin (which may be endogenous or be present as a transgene)and/or for altering activity of the haemoglobin encoded by this nucleicacid sequence.

Therefore, according to one aspect of the present invention, there isprovided a method for modifying the growth characteristics of a plant,comprising exogenous application of one or more compounds or elementscapable of altering expression of a haemoglobin gene and/or capable ofaltering activity of a haemoglobin protein.

Additionally or alternatively, and according to a preferred embodimentof the present invention, altering expression of a nucleic acid sequenceencoding a haemoglobin and/or altering activity of the haemoglobinitself may be effected by recombinant means. Such recombinant means maycomprise a direct and/or indirect approach for altering of expression ofa nucleic acid sequence and/or for altering of the activity of aprotein.

For example, an indirect approach may comprise introducing, into aplant, a nucleic acid sequence capable of altering activity of theprotein in question (a haemoglobin) and/or expression of the gene inquestion (a gene encoding a haemoglobin). The haemoglobin gene or thehaemoglobin protein may be wild type, i.e. the native or endogenousnucleic acid or polypeptide. Alternatively, it may be a nucleic acidderived from the same or another species, which gene is introduced as atransgene, for example by transformation. This transgene may besubstantially modified from its native form in composition and/orgenomic environment through deliberate human manipulation. Alsoencompassed by an indirect approach for altering activity of anhaemoglobin and/or expression of a haemoglobin gene is the inhibition orstimulation of regulatory sequences that drive expression of the nativegene or transgene. Such regulatory sequences may be introduced into aplant.

A direct and preferred approach on the other hand comprises introducinginto a plant a nucleic add sequence encoding a haemoglobin or ahomologue, derivative or active fragment thereof. The nucleic acidsequence may be introduced into a plant by, for example, transformation.The nucleic acid sequence may be derived (either directly or indirectly(if subsequently modified)) from any source provided that the sequence,when expressed in a plant, leads to altered expression of a haemoglobinnucleic acid/gene or altered activity of a haemoglobin protein. Thenucleic acid sequence may be isolated from a microbial source, such asbacteria, yeast or fungi, or from a plant, algal or animal (includinghuman) source. This nucleic acid may be substantially modified from itsnative form in composition and/or genomic environment through deliberatehuman manipulation. The nucleic acid sequence is preferably a homologousnucleic acid sequence, i.e. a nucleic acid sequence obtained from aplant, whether from the same plant species or different.

Another embodiment of the present invention provides a nucleic acidconstruct comprising a nucleic acid sequence according to the invention,as described above. The nucleic add construct may be an expressionvector, wherein the nucleic acid sequence is operably linked to one ormore control sequences allowing expression of the sequence inprokaryotic and/or eukaryotic host cells.

Thus, according to the present invention, there is provided a nucleicacid construct, comprising:

-   -   (i) an isolated nucleic acid sequence according to any of (i)        to (viii) as defined above; and    -   (ii) one or more control sequences controlling expression of the        nucleic acid sequence of (i); and optionally,    -   (iii) a transcription terminator sequence.

Preferably, the isolated nucleic acid sequence (i) of the nucleic acidconstruct above is as represented by SEQ ID NO 1. To effect expressionof a protein in a cell, tissue or organ, preferably of plant origin,either the protein may be introduced directly to a cell, such as bymicroinjection or ballistic means or alternatively, an isolated nucleicacid molecule encoding the protein may be introduced into a cell, tissueor organ in an expressible format. Preferably, the DNA sequence of theinvention comprises a coding sequence or open reading frame (ORF)encoding the polypeptide of the invention or a homologue or derivativethereof or an immunologically active fragment thereof as defined supra.

With “vector” or “vector sequence” is meant a DNA sequence, which may beintroduced in an organism by transformation and which may be stablymaintained in that organism. Vector maintenance is possible in e.g.cultures of Escherichia coli, Agrobacterium tumefaciens, Saccharomycescerevisiae or Schizosaccharomyces pombe. Other vectors such as phagemidsand cosmid vectors may be maintained and multiplied in bacteria and/orviruses. Vector sequences generally comprise a set of unique sitesrecognised by restriction enzymes, the multiple cloning site (MCS), inwhich one or more inserts may be inserted. With “insert” is meant a DNAsequence which is integrated in one or more of the sites of the MCScomprised within a vector. “Expression vectors” form a subset of vectorswhich comprise regulatory sequences enabling expression of the proteinencoded by (an) insert(s). Expression vectors are known in the artenabling protein expression in organisms including bacteria (e.g. E.coli), fungi (e.g. S. cerevisiae, S. pombe, P. pastoris), insect cells(e.g. baculoviral expression vectors), animal cells (e.g. COS or CHOcells) and plant cells (e.g. potato virus X-based expression vectors,see e.g. Vance et al. 1998—WO 98/44097). The invention includes anyvector or expression vector comprising an insert encoding a proteinaccording to the invention, homologues, derivatives, functional and/orimmunologically active fragments thereof as defined supra.

By “expressible format” is meant that the isolated nucleic add moleculeis in a form suitable for being transcribed into mRNA and/or translatedto produce a protein, ether constitutively or following induction by anintracellular or extracellular signal, such as an environmental stimulusor stress (mitogens, anoxia, hypoxia, temperature, salt, light,dehydration, etc.) or a chemical compound such as IPTG(isopropyl-β-D-thiogalactopyranoside) or such as an antibiotic(tetracycline, ampicillin, rifampicin, kanamycin), hormone (e.g.gibberellin, auxin, cytokinin, glucocorticoid, brassinosteroid,ethylene, abscisic acid etc), hormone analogue (iodoacetic acid (IAA),2,4-D, etc), metal (zinc, copper, iron, etc), or dexamethasone, amongstothers. As will be known to those skilled in the art, expression of afunctional protein may also require one or more post-translationalmodifications, such as glycosylation, phosphorylation,dephosphorylation, or one or more protein-protein interactions, amongstothers. All such processes are included within the scope of the term“expressible format”.

“Expression” means the production of a protein or nucleotide sequence inthe cell itself or in a cell-free system. It includes transcription intoan RNA product, post-transcriptional modification and/or translation toa protein product or polypeptide from a DNA encoding that product, aswell as possible post-translational modifications. Preferably,expression of a protein in a specific cell, tissue, or organ, preferablyof plant origin, is effected by introducing and expressing an isolatednucleic add molecule encoding the protein, such as a cDNA molecule,genomic gene, synthetic oligonucleotide molecule, mRNA molecule or openreading frame, to the cell, tissue or organ, wherein the nucleic addmolecule is placed in operable connection with suitable regulatorysequences including a promoter, preferably a plant-expressible promoter,and optionally a terminator sequence.

“Regulatory sequence” refers to control DNA sequences, which arenecessary to affect the expression of coding sequences to which they areligated and the stability of the transcription products resulting fromthese coding sequences. The nature of such control sequences differsdepending upon the host organism. In prokaryotes, control sequencesgenerally include promoters, ribosomal binding sites, and terminators.In eukaryotes, control sequences generally include promoters,terminators and enhancers or silencers. The term “control sequence” isintended to include, as a minimum, all components necessary forexpression and may also include additional advantageous components whichdetermine when, how much and where a specific gene is expressed, as wellas influence the stability of transcripts. Reference herein to a“promoter” is to be taken in its broadest context and includes thetranscriptional regulatory sequences derived from a classical eukaryoticgenomic gene, including the TATA box which is required for accuratetranscription initiation, with or without a CCAAT box sequence andadditional regulatory elements (i.e. upstream activating sequences,enhancers and silencers) which after gene expression in response todevelopmental and/or external stimuli, or in a tissue-specific manner.

As used herein, the term “derived from” or “originated from” shall betaken to indicate that a particular integer or group of integersoriginates from the species specified but has not necessarily beenobtained directly from the specified source.

Regulatory sequences herein also refer to any of the group comprising apromoter, enhancer, silencer, intron sequence, 3′UTR and/or 5′UTRregion, protein and/or RNA stabilizing elements. The term “promoter”also includes the transcriptional regulatory sequences of a classicalprokaryotic gene, in which case it may include a −35 box sequence and/ora −10 box transcriptional regulatory sequences. The term “promoter” isalso used to describe a synthetic or fusion molecule or derivative,which confers, activates or enhances expression of a nucleic addmolecule in a cell, tissue or organ. Promoters may contain additionalcopies of one or more specific regulatory elements, to further enhanceexpression and/or to alter the spatial expression and/or temporalexpression of a nucleic acid molecule to which it is operably connected.Such regulatory elements may be placed adjacent to a heterologouspromoter sequence to drive expression of a nucleic acid molecule inresponse to external stimuli or to confer expression of a nucleic acidmolecule to specific cells, tissues or organs such as meristems, leaves,roots, embryo, flowers, seeds or fruits.

In the context of the present invention, the promoter is preferably aplant-expressible promoter sequence. Promoters, however, that alsofunction or solely function in non-plant cells such as bacteria, yeastcells, insect cells and animal cells are not excluded from theinvention, for example where the methods of the invention are applied inmodifying stress tolerance of yeast. By “plant-expressible” is meantthat the promoter sequence, including any additional regulatory elementsadded thereto or contained therein, is at least capable of inducing,conferring, activating or enhancing expression in a plant cell, tissueor organ, preferably a monocotyledonous or dicotyledonous plant cell,tissue, or organ. In the present context, a “regulated promoter” or“regulatable promoter sequence” is a promoter that is capable ofconferring expression on a structural gene in a particular cell, tissueor organ, or group of cells, tissues or organs of a plant, optionallyunder specific conditions, however, it generally does not conferexpression throughout the plant under all conditions. Accordingly, aregulatable promoter sequence may be a promoter sequence that drivesexpression of a gene to which it is operably connected in a particularlocation within the plant or alternatively throughout the plant under aspecific set of conditions, such as following induction of geneexpression by a chemical compound or other elicitor.

A regulatable promoter that may be used in the performance of thepresent invention confers expression in a specific location within theplant and/or a specific developmental phase of a plant, eitherconstitutively or following induction. Included within the scope of suchpromoters are cell-specific promoter sequences, tissue-specific promotersequences, organ-specific promoter sequences, cell cycle specific genepromoter sequences, inducible promoter sequences and constitutivepromoter sequences that have been modified to confer expression in aparticular part of the plant at any one time, such as by integration ofa constitutive promoter within a transposable genetic element (Ac, Ds,Spm, En, or other transposon). Those skilled in the art will be awarethat an “inducible promoter” is a promoter, the transcriptional activityof which is increased or induced in response to a developmental,chemical, environmental, or physical stimulus. Similarly, the skilledcraftsman will understand that a “constitutive promoter” is a promoterthat is transcriptionally active throughout most, but not necessarilyall phases of growth and development of an organism, preferably a plant.In contrast, the term “ubiquitous promoter” is taken to indicate apromoter that is transcriptionally active throughout most, but notnecessarily all parts of an organism, preferably a plant.

The term “cell-specific” shall be taken to indicate that expression ispredominantly in a particular cell or cell-type, preferably of plantorigin, albeit not necessarily exclusively in that cell or cell-type.Similarly, the term “tissue-specific” shall be taken to indicate thatexpression is predominantly in a particular tissue or tissue-type,preferably of plant origin, albeit not necessarily exclusively in thattissue or tissue-type. Similarly, the term “organ-specific” shall betaken to indicate that expression is predominantly in a particularorgan, preferably of plant origin, albeit not necessarily exclusively inthat organ.

Constitutive promoters or promoters that induce expression throughoutthe entire plant may be modified by the addition of nucleotide sequencesderived from one or more tissue-specific promoters or tissue-specificinducible promoters, to confer tissue-specificity thereon. For example,the CaMV 35S promoter may be modified by the addition of maize Adh1promoter sequence, to confer anaerobically-regulated root-specificexpression thereon (Ellis et al., 1987). Another example includesconferring root specific or root abundant gene expression by fusing theCaMV35S promoter to elements of the maize glycine-rich protein GRP3 gene(Feix and Wulff 2000—WO0015662). Those skilled in the art will readilybe capable of selecting appropriate promoter sequences frompublicly-available or readily-available sources, for use in regulatingexpression of the polypeptides described supra.

A preferred promoter according to the invention would be a constitutivepromoter such as GOS2 or CaMV 35S, or a promoter inducible byenvironmental stimuli, or a seed specific promoter.

Placing a nucleic acid molecule under the regulatory control of apromoter sequence, or in operable connection with a promoter sequencemeans positioning the nucleic acid molecule such that expression iscontrolled by the promoter sequence. A promoter is usually, but notnecessarily, positioned upstream, or at the 5′-end, and within 2 kb ofthe start site of transcription of the nucleic acid molecule which itregulates. In the construction of heterologous promoter/structural genecombinations it is generally preferred to position the promoter at adistance from the gene transcription start site that is approximatelythe same as the distance between that promoter and the gene it controlsin its natural setting (i.e., the gene from which the promoter isderived). As is known in the art, some variation in this distance may beaccommodated without loss of promoter function. Similarly, the preferredpositioning of a regulatory sequence element with respect to aheterologous gene to be placed under its control is defined by thepositioning of the element in its natural setting (i.e., the gene fromwhich it is derived). Again, as is known in the art, some variation inthis distance may also occur.

“Operably linked” refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. A control sequence “operably linked” to a codingsequence is ligated in such a way that expression of the coding sequenceis achieved under conditions compatible with the control sequences.Where the control sequence is a promoter, it would be obvious for askilled person to use a double-stranded nucleic acid.

The term “terminator” refers to a DNA sequence at the end of atranscriptional unit which signals termination of transcription.Terminators are 3′-non-translated DNA sequences containing apolyadenylation signal, which facilitates the addition of polyadenylatesequences to the 3′-end of a primary transcript. Terminators active incells derived from viruses, yeasts, moulds, bacteria, insects, birds,mammals and plants are known and described in the literature. They maybe isolated from bacteria, fungi, viruses, animals and/or plants.

Examples of terminators particularly suitable for use in the geneconstructs of the present invention include the Agrobacteriumtumefaciens nopaline synthase (NOS) gene terminator, the Agrobacteriumtumefaciens octopine synthase (OCS) gene terminator sequence, theCauliflower mosaic virus (CaMV) 35S gene terminator sequence, the Oryzasativa ADP-glucose pyrophosphorylase terminator sequence (t3′Bt2), theZea mays zein gene terminator sequence, the rbcs-1A gene terminator, andthe rbcs-3A gene terminator sequences, amongst others.

The nucleic acid constructs of the invention may further include anorigin of replication sequence which is required for maintenance and/orreplication in a specific cell type, for example a bacterial cell, whensaid nucleic acid construct is required to be maintained as an episomalgenetic element (e.g. plasmid or cosmid molecule) in a cell. Preferredorigins of replication include, but are not limited to, the f1-ori andcolE1 origins of replication.

The nucleic acid construct may optionally comprise a selectable markergene. As used herein, the term “selectable marker gene” includes anygene which confers a phenotype on a cell in which it is expressed tofacilitate the identification and/or selection of cells which aretransfected or transformed with a nucleic acid construct of theinvention or a derivative thereof. Suitable markers may be selected frommarkers that confer antibiotic or herbicide resistance. Cells containingthe recombinant DNA will thus be able to survive in the presence ofantibiotic or herbicide concentrations that kill untransformed cells.Examples of selectable marker genes include the bar gene which providesresistance to the herbicide Basta; the nptII gene which confersresistance to the antibiotic kanamycin and neomycin; the ampicillinresistance (Amp′), tetracycline resistance gene (Tc′), the hpt genewhich confers hygromycin resistance, the phosphinothricin resistancegene, chloramphenicol acetyltransferase (CAT) gene, the hygromycinresistance gene. Visual markers, such as the Green Fluorescent Protein(GFP), β-glucuronidase (GUS) gene, and luciferase gene, amongst othersmay also be used as selectable markers.

Recombinant DNA constructs for use in the methods according to thepresent invention may be constructed using recombinant DNA technologywell known to persons skilled in the art. The gene constructs may beinserted into vectors, which may be commercially, suitable fortransforming into host cells, preferably plant cells, and suitable forexpression of the gene of interest in the transformed cells.

According to a preferred feature of the present invention, the nucleicacid sequence encoding haemoglobin is overexpressed in a plant. Methodsfor obtaining enhanced or increased expression of genes or gene productsare well documented in the art and include, for example, overexpressiondriven by a strong promoter, the use of transcription enhancers ortranslation enhancers.

On the other hand, downregulation of the nucleic acid sequence may alsogive rise to altered growth characteristics in a plant. Plants havingmodified growth characteristics may be obtained by expressing a nucleicacid sequence encoding haemoglobin in either sense or antisenseorientation. Techniques for downregulation are well known in the art.“Gene silencing” or “downregulation” of expression, as used herein,refers to lowering levels of gene expression and/or levels of activegene product and/or levels of gene product activity. Such decreases inexpression may be accomplished by, for example, the addition of codingsequences or parts thereof in a sense orientation (if it is desired toachieve co-suppression).

Nucleic acid constructs (genetic constructs) aimed at silencing geneexpression may comprise the nucleotide sequence encoding a haemoglobin,for example the sequence itself as represented by SEQ ID NO 1 (or one ormore parts thereof) in a sense and/or antisense orientation relative tothe promoter sequence. The sense or antisense copies of at least part ofthe endogenous gene in the form of direct or inverted repeats may beutilized in the methods according to the invention. The characteristicsof plants may also be altered by introducing into a plant at least partof an antisense version of the nucleotide sequence represented by SEQ IDNO 1. It should be clear that part of the nucleic acid could achieve thedesired result.

Homologous anti-sense genes are preferred to heterologous anti-sensegenes, homologous genes being plant genes, preferably plant genes fromthe same plant species, and heterologous genes being genes fromnon-plant species.

Another method for downregulation of gene expression or gene silencingcomprises use of ribozymes, for example as described in Atkins et al.1994 (WO 94/00012), Lenee et al. 1995 (WO 95/03404), Lutziger et al.2000 (WO 00/00619), Prinsen et al. 1997 (WO 97/3865) and Scott et al.1997 (WO 97/38116).

Gene silencing may also be achieved by insertion mutagenesis (forexample, T-DNA insertion or transposon insertion), or by gene silencingstrategies, including RNA mediated silencing, as described by, amongothers, Angell and Baulcombe 1998 (WO 98/36083), Lowe et al. 1989 (WO98/53083), Lederer et al. 1999 (WO 99/15682) or Wang et al. 1999 (WO99/53050). Expression of an endogenous gene essentially similar to SEQID NO 1, 3, or 5, or the activity of the encoded protein may also bereduced if there is a mutation on the endogenous gene.

Furthermore, the present invention also relates to a method for theproduction of a transgenic plant having altered growth characteristicswhen compared to wild type plants, comprising the steps of:

-   -   (i) introducing into a plant or plant cell a nucleic acid        sequence encoding a haemoglobin according to the present        invention; and    -   (ii) cultivating this plant or plant cell under conditions        promoting regeneration and/or mature plant growth.

The term “plant cell” comprises any cell derived from any plant andexisting in culture as a single cell, a group of cells or a callus. Aplant cell may also be any cell in a developing or mature plant inculture or growing in nature.

Advantageously, the methods of the invention are applicable to anyplant. “Plant” or “Plants” comprise all plant species which belong tothe superfamily Viridiplantae. The present invention is applicable toany plant, in particular monocotyledonous plants and dicotyledonousplants including a fodder or forage legume, ornamental plant, food crop,tree, or shrub selected from the list comprising Acacia spp., Acer spp.,Actinidia spp., Aesculus spp., Agathis australis, Albizia amara,Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Asteliafragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassicaspp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadabafarinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicumspp., Cassia spp., Centroema pubescens, Chaenomeles spp., Cinnamomumcassia, Coffea arabica, Colophospermum mopane, Coronillia varia,Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp.,Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogonspp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davalliadivaricata, Desmodium spp., Dicksonia squarosa, Diheteropogonamplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloapyramidalis, Ehrartia spp., Eleusine coracana, Eragrestis spp.,Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulalia villosa,Fagopyrum spp., Feijoa sellowiana, Fragaria spp., Flemingia spp,Freycinetia banksii, Geranium thunbergii, Ginkgo biloba, Glycinejavanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtiacoleosperma, Hedysarum spp., Hemarthia altissima, Heteropogon contortus,Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypertheliadissoluta, Indigo incarnate, Iris spp., Leptarrhena pyrolifolia,Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex,Lotonus bainesii, Lotus spp., Macrotyloma axillare, Malus spp., Manihotesculenta, Medicago sativa, Metasequoia glyptostroboides, Musasapientum, Nicotianum spp., Onobrychis spp., Omithopus spp., Oryza spp.,Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp.,Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp.,Picea glauca, Pinus spp., Pisum sativum, Podocarpus totara, Pogonarthriafleckii, Pogonarthria squarrosa, Populus spp., Prosopis cineraria,Pseudotsuge menziesii, Pterolobium stellatum, Pyrus communis, Quercusspp., Rhaphiolepsis umbellate, Rhopalostylis sapida, Rhus natalensis,Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubusspp., Salix spp., Schyzachyrium sanguineum, Sciadopitys verticillate,Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor,Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides,Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themedatriandra, Trifolium spp., Triticum spp., Tsuga heterophylia, Vacciniumspp., Vicia spp. Vitis vinifera, Watsonia pyramidata, Zantedeschiaaethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brusselssprouts, cabbage, canola, carrot, cauliflower, celery, collard greens,flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean,straw, sugar beet, sugar cane, sunflower, tomato, squash, and tea,amongst others, or the seeds of any plant specifically named above or atissue, cell or organ culture of any of the above species. According toa preferred feature of the present invention, the plant is a crop plantcomprising soybean, sunflower, canola, alfalfa, rapeseed or cotton.Further preferably, the plant according to the present invention is amonocotyledonous plant including members of the Poaceae, such assugarcane, most preferably the plant is a cereal, such as rice, maize,wheat, millet, barley and sorghum, oats.

The present invention clearly extends to any plant cell or plantobtainable by any of the methods described herein, and to all plantparts and propagules thereof. The invention comprises any plant cell,plant part or plant having altered characteristics as described above,including increased yield, increased biomass, increased cell division,increased tolerance to osmotic stress and/or altered architecture, saidplant cell, plant part or plant having increased expression of a nucleicacid sequence encoding a plant class-2 non-symbiotic haemoglobin and/orhaving altered activity of a plant class-2 non-symbiotic haemoglobinprotein. The invention also comprises any plant cell, plant part orplant having increased tolerance to high temperature stress said plantcell, plant part or plant having increased expression of a nucleic acidsequence encoding a plant haemoglobin protein. Also transgenicharvestable parts or propagules of such plants are encompassed by thepresent invention, wherein the harvestable parts are selected from thegroup consisting of seeds, leaves, flowers, fruits, stem cultures,rhizomes, tubers and bulbs. The term plant furthermore encompassessuspension cultures, embryos, meristematic regions, callus tissue,leaves, seeds, roots, shoots, gametophytes, sporophytes, pollen, andmicrospores.

The present invention extends further to encompass the transgenicancestors or progeny of a transformed or transfected cell, tissue, organor whole plant that has been produced by any of the aforementionedmethods, the only requirement being that progeny exhibit the samegenotypic and/or phenotypic characteristic(s) as those produced in theparent by the methods according to the invention.

The nucleic acid molecule or a nucleic acid construct comprising it maybe introduced into a cell using any known method for the transfection ortransformation of a cell. A whole organism may be regenerated from asingle transformed or transfected cell, using methods known in the art.Plant tissue capable of subsequent clonal propagation, whether byorganogenesis or embryogenesis, may be transformed with a nucleic acidconstruct of the present invention and a whole plant regeneratedtherefrom. The particular tissue chosen will vary depending on theclonal propagation systems available for, and best suited to, theparticular species being transformed. Exemplary tissue targets includeleaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes,callus tissue, existing meristematic tissue (e.g., apical meristem,axillary buds, and root meristems), and induced meristem tissue (e.g.,cotyledon meristem and hypocotyl meristem).

The gene of interest is preferably introduced into a plant bytransformation. The term “transformation” as referred to hereinencompasses the transfer of an exogenous polynucleotide into a hostcell, irrespective of the method used for transfer. The polynucleotidemay be transiently or stably introduced into a host cell and may bemaintained non-integrated, for example, as a plasmid, or alternatively,may be integrated into the host genome. The resulting transformed plantcell may then be used to regenerate a transformed plant in a mannerknown to persons skilled in the art. Transformation of a plant speciesis now a fairly routine technique.

Transformation methods include the use of liposomes, electroporation,chemicals that increase free DNA uptake, injection of the DNA directlyinto the plant, particle gun bombardment, transformation using virusesor pollen and microprojection. Methods may be selected from thecalcium/polyethylene glycol method for protoplasts (Krens, F. A. et al.,1882, Nature 296, 72-74; Negrutiu I. et al., 1987, Plant Mol. Biol. 8,363-373); electroporation of protoplasts (Shillito R. D. et al., 1985Bio/Technol 3, 1099-1102); microinjection into plant material (CrosswayA. et al., 1986, Mol. Gen Genet 202, 179-185); DNA or RNA-coatedparticle bombardment (Klein T. M. et al., 1987, Nature 327, 70)infection with (non-integrative) viruses and the like. A preferredmethod according to the present invention is Agrobacterium-mediatedtransformation (An et al., EMBO J., 4, 277-284, 1985; Dodds, Plantgenetic engineering, 1985; Herrera-Estrella et al., EMBO J., 2, 987-995,1983; Herrera-Estrella et al., Nature, 303, 209-213, 1983), includingthe ‘flower dip’ transformation method; (Bechtold and Pelletier, MethodsMol. Biol., 82, 259-266, 1998; Trieu et al., Plant J., 22 (6), 531-541,2000).

A whole plant may be regenerated from the transformed or transfectedcell, in accordance with procedures well known in the art. Plant tissuecapable of subsequent clonal propagation, whether by organogenesis orembryogenesis, may be transformed with a gene construct of the presentinvention and a whole plant regenerated therefrom. The particular tissuechosen will vary depending on the clonal propagation systems availablefor, and best suited to, the particular species being transformed.Exemplary tissue targets include leaf disks, pollen, embryos,cotyledons, hypocotyls, megagametophytes, callus tissue, existingmeristematic tissue (e.g., apical meristem, axillary buds, and rootmeristems), and induced meristem tissue (e.g., cotyledon meristem andhypocotyl meristem). The term “organogenesis”, as used herein, means aprocess by which shoots and roots are developed sequentially frommeristematic centers. The term “embryogenesis”, as used herein, means aprocess by which shoots and roots develop together in a concertedfashion (not sequentially), whether from somatic cells or gametes.

Generally after transformation, plant cells or cell groupings areselected for the presence of one or more markers which are encoded byplant-expressible genes co-transferred with the gene of interest,following which the transformed material is regenerated into a wholeplant. Putatively transformed plants may be evaluated, for instanceusing Southern analysis, for the presence of the gene of interest, copynumber and/or genomic organisation. Alternatively or additionally,expression levels of the newly introduced DNA may be undertaken usingNorthern and/or Western analysis, both techniques being well known topersons having ordinary skill in the art.

The generated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedto give homozygous second generation (or T2) transformants, and the T2plants further propagated through classical breeding techniques.

The generated transformed organisms may take a variety of forms. Forexample, they may be chimeras of transformed cells and non-transformedcells; clonal transformants (e.g., all cells transformed to contain theexpression cassette); grafts of transformed and untransformed tissues(e.g., in plants, a transformed rootstock grafted to an untransformedscion).

The present invention also relates to the use of a plant non-symbiotichaemoglobin or of a nucleic acid sequence encoding a plant non-symbiotichaemoglobin for altering characteristics of a plant.

In particular, the present invention relates to the use of a nucleicacid sequence encoding plant class-2 non-symbiotic haemoglobin forincreasing one or more of yield, biomass or cell division of a plant.Preferably, the increased yield comprises at least increased seed yield.

The present invention furthermore relates to the use of a nucleic acidsequence encoding plant class-2 non-symbiotic haemoglobin for alteringarchitecture of a plant.

Preferably, the nucleic acid sequence encoding plant class-2non-symbiotic haemoglobin used for increasing one or more of yield,biomass or cell division of a plant or for altering architecture of aplant is isolated from a dicotyledonous plant, preferably fromBrassicaceae, more preferably from Arabidopsis thaliana, most preferablythe isolated nucleic acid is as represented by SEQ ID NO 3.

The present invention also relates to the use of a nucleic acid sequenceencoding plant class-2 non-symbiotic haemoglobin for increasing abioticstress tolerance of a plant. Preferably the abiotic stress is osmoticstress. The present invention also relates to the use of a nucleic acidsequence encoding plant haemoglobin for increasing high temperaturetolerance of a plant, preferably said plant haemoglobin is anon-symbiotic haemoglobin, more preferably a class-2 non-symbiotichaemoglobin.

Preferably, the nucleic acid sequence encoding plant class-2non-symbiotic haemoglobin for increasing osmotic stress tolerance or forincreasing high temperature stress tolerance is isolated from adicotyledonous plant, preferably from the Brassicaceae, more preferablyfrom Beta vulgaris, most preferably the isolated nucleic acid isessentially similar to SEQ ID NO 1.

Furthermore, the present invention also relates to the use of a nucleicacid sequence encoding a haemoglobin and to the use of a haemoglobinitself for altering stress tolerance of bacteria or yeast. The inventionfurther also extends to the use of a nucleic acid sequence encodinghaemoglobin according to the present invention and homologues,derivatives and active fragments thereof and to the use of thehaemoglobin itself and of homologues, derivatives and active fragmentsthereof in therapeutic or diagnostic compositions. The invention alsoextends to the use of a nucleic acid sequence encoding plant class-2non-symbiotic haemoglobin according to the invention and homologues,derivatives and active fragments thereof and to the use of thehaemoglobin itself and of homologues, derivatives and active fragmentsthereof in modulating levels of O₂ or other compounds, such as, forexample, NO. In this respect, modulating levels of a plant class-2non-symbiotic haemoglobin according to the invention may also be used tomodify existing signal transduction pathways in organisms. Therefore thepresent invention also provides the use of a nucleic acid sequenceencoding a plant class-2 non-symbiotic haemoglobin according to theinvention and/or of the haemoglobin itself in modifying signaltransduction pathways. These uses are also encompassed by the presentinvention.

The nucleic acid sequences hereinbefore described (and portions of thesame and sequences capable of hybridising with the same) and the aminoacid sequences hereinbefore described (and homologues, derivatives andactive fragments of the same) are useful in modifying the growthcharacteristics of plants, as hereinbefore described. The sequenceswould therefore find use as growth regulators, such as herbicides orgrowth stimulators. The present invention also provides a compositioncomprising a protein represented by any of the aforementioned amino acidsequences or homologues, derivatives or active fragments thereof for useas a growth regulator.

DESCRIPTION OF FIGURES

FIG. 1: Pileup and unrooted dendrogram showing homology betweenhaemoglobin sequences from Arabidopsis thaliana (at, SEQ ID NO:4),Brassica napus (bn, SEQ ID NO:6), Beta vulgaris (bv, SEQ ID NO:2),Gossypium hirsutum (gh, SEQ ID NO:19), Lycopersicon esculentum (le, SEQID NO:20), Casuarina glauca (cg, SEQ ID NO:21).

FIG. 2: The BvXero2 gene confers tolerance to both osmotic stress andhigh temperature in yeast. The upper row represents the wild-type yeast,the bottom row is the yeast strain transformed with BvXero2. From leftto right: control on YPD, growth at 37° C. after 2 and 3 days, andgrowth on 1.7 M sorbitol after 4 days.

FIG. 3: Southern blot with BvXero2 as probe on genomic sugar beet DNA.Enzymes used were BamHI, HindIII and EcoRI. At the right 1 kb markersare depicted.

FIG. 4: Northern blot with BvXero2 as probe. Different time points (inhrs) after treating the sugar beet plants with 250 mM NaCl. α₃-tubulinwas used as control.

FIG. 5: Northern blot with BvXero2 as probe. Different time points (inhrs) after treating the sugar beet plants with 100 μM ABA α₃-tubulin wasused as control.

FIG. 6: Schematic representation of the entry done p55, containingCDS2591 within the AttL1 and AttL2 sites for Gateway® cloning in thepDONR201 backbone. CDS2591 is the internal code for the Arabidopsisnon-symbiotic haemoglobin Hb2. This vector contains also a bacterialkanamycin-resistance cassette and a bacterial origin of replication.

FIG. 7: Binary vector for the expression in plants of CDS2591 under thecontrol of P35 S and the T-zein-T-rbcS-deltaGA double terminatorsequence. CDS2195 is the internal code for Arabidopsis non-symbiotichaemoglobin Hb2. This vector contains a T-DNA derived from the TiPlasmid, limited by a left border (LB repeat, LB Ti C58) and a rightborder (RB repeat, RB Ti C58)). From the left border to the rightborder, this T-DNA contains: a selectable marker cassette for antibioticselection of transformed plants; a screenable marker cassette for visualscreening of transformed plants; the ‘constitutive promoter—CDS2591—zeinand rbcS-deltaGA double terminator’. This vector also contains an originof replication from pBR322 for bacterial replication and a selectablemarker (Spe/SmeR) for bacterial selection with spectinomycin andstreptomycin.

FIG. 8: Rossette Area. Average rosette area for transgenic and controlnon-transgenic plants is represented in arbitrary units at 4 time pointsbetween 21 and 45 dpi. Standard error bars are shown.

FIG. 9: Transgenic plants (2 plants on the left) and non-transgeniccontrol plants (2 plants in the right) 5 weeks upon recovery from stress(150 mM NaCl). Picture was taken at 54 dpi.

FIG. 10: Sequence listing.

EXAMPLES

The present invention will now be described with reference to thefollowing examples, which are by way of illustration alone.

Unless stated otherwise in the Examples, all recombinant DNA techniquesare performed according to protocols as described in Sambrook et al.(1989) or in Volumes 1 and 2 of Ausubel et al. (2000). Standardmaterials and methods for plant molecular work are described in R. D. D.Croy (1993).

Example 1 Construction of a Sugar Beet cDNA Library Induced by SaltStress

Sugar beet seeds (Beta vulgaris cv. Dita) were sown on pots containing amixture of sand and vermiculite (1:1 w/w). The plants were grown undergreenhouse conditions (8 h at 20° C., 16 h at 25° C. with supplementarylighting to simulate a minimum 12 h photoperiod). The plants wereperiodically irrigated with a nutrient solution containing 2.4 g/lCa(NO₃)₂.4H₂O, 1 g/l KNO₃, 1 g/l MgSO₄.7H₂O, 0.3 g/l KH₂PO₄, 5.6 mg/lFequelate (Kelantren, Bayer), 1.1 mg/l ZnSO₄.7H₂O, 3.3 mg/l MnO₄.H₂O,0.3 mg/l CuSO₄.5H₂O, 3.8 mg/l H₃BO₃, 0.18 mg/l (NH₄)₆Mo₇.4H₂O. For theconstruction of the cDNA library, 3-week old plants were irrigated with200 mM NaCl the day preceding the harvesting.

Directional cDNA synthesis was performed with the cDNA Synthesis Kit(Stratagene) using poly(A)⁺ RNA prepared from leaves of salt-treatedsugar beet plants. The cDNA was ligated into the phage λPG15 vector andpacked using Gigapack III Gold Packaging Extract (Stratagene). Thisphage carries the excisable expression plasmid pYPGE15 (URA3 as aselection marker) that may be used directly for both E. coli and yeastcomplementation (Brunelli and Pall, Yeast, 9, 1309-1318, 1993). Aplasmid cDNA library was recovered from λPG15 with the cre-loxrecombinase system (Brunelli and Pall, Yeast, 9, 1309-1318, 1993).

Example 2 Set-Up of a Screening Assay for Osmotic Stress Tolerance

The yeast strains used were the diploid strain W303/W303(can1-100,his3-11,15,leu2-3,112, trp1-1,ura3-1,GAL+) and a diploid mutant thereof,deficient for glycerol phosphate dehydrogenase (gpd1), named JM164,constructed from two haploid gpd1 mutant strains (YRA111(W303-1Agpd1::TRP1 mat a) and YRA114 (W303-1A gpd1::TRP1 mat α)). The diploidstrains were used because these prevent the isolation of recessivechromosomal mutations which might give tolerance to osmotic stress. Thestrains were grown on YPD medium (2% glucose, 2% peptone and 1% yeastextract) or on SD medium (2% glucose, 0.7% yeast nitrogen base (Difco)without amino acids, 50 mM MES [2-(N-morpholino)ethanesulfonic acid]adjusted to pH 5.5 with Tris (Tris(hydroximethyl) aminomethane), and therequired amino acids, purine and pyrimidine bases).

In a first step, the sensitivity to sorbitol of a gpd1 mutant strain(JM164) was compared with that of a wild type diploid strain, in bothYPD and SD medium. To this end, the yeast strains were grown on YPD oron SD medium with different concentrations of sorbitol, ranging from 1.3M to 1.8 M at a temperature of 28° C. for 4 days. At 1.7 M sorbitol, aclear difference in growth was observed between the gpd1 mutant and thewild type. The gpd1 mutant strain was more sensitive compared to thewild type.

In a second step, the best conditions for the transformation weredetermined, optimising the amount of cells and the amount of libraryplasmid to be used in a transformation reaction. 300 ml of YPD wasinoculated with 30 μl of a saturated preculture of JM164 cells. Thisculture was grown overnight until an OD₆₆₀≅0.8 was obtained. The yeastcells were centrifuged at 2000 rpm, washed with water and then washedwith AcLiTE solution (0.1 M lithium acetate, 10 mM Tris-HCl pH 7.6 and 1mM EDTA (Ethylene diamine tetraacetic acid, disodium salt)). The pelletof cells was resuspended in 2 ml of AcLiTE solution and incubated for 15minutes with shaking at 300° C. After incubation, 200 μl of ssDNA (10mg/ml) was added. The solution was then divided into 110 μl aliquotswhich were placed in an Eppendorf tube, and 200 ng of cDNA library wereadded. This was followed by heat shock transformation using the methoddescribed by Gietz et al in brief, 500 μl of PEG-AcLiTE solution (AcLiTEsolution with 40% w/w of PEG (Polyethylene glycol) 4000) was added toeach aliquot. After shaking, aliquots were incubated for 30 minutes at300° C. and for twenty minutes at 42° C. The cells were then harvestedand resuspended in 200 μl of 1M sorbitol. Two aliquots were plated onto14 cm Ø Petri dishes containing SD with all the necessary supplementsexcept tryptophan (marker for the gpd1 mutation), and uracil (marker forthe plasmid). To quantify the efficiency of the transformation, four 55μl aliquots were separated from the original cell pellet and inoculatedwith 0, 10, 50 and 100 ng of cDNA library. The same transformationprotocol was then applied, and, at the end the cells were resuspended in100 μl of sorbitol and plated onto a 7 cm Ø Petri dish containing thesame SD solution. The average transformation efficiency for the JM164strain was about 20 transformants for each ng of cDNA library.

Example 3 Isolation of Xero Genes

Three days after transformation, colonies had developed. The colonieswere harvested in sterile water and the number of cells was quantifiedby plating different dilutions. The cell suspension obtained afterharvesting was concentrated about ton times and was plated on YPD mediumor SD medium containing 1.7 M sorbitol. The plates were incubated at 28°C. and colonies able to grow after four days were selected. Thetolerance of the colonies isolated in the first round was re-checked onselective medium and those clones not giving significant tolerance werediscarded. From the colonies that remained, the plasmid was eliminatedby selection in minimal medium. This was done by obtaining stationaryphase cultures of each strain in liquid YPD medium. These cultures wereplated in YPD medium and after two days colonies were picked andreplicated both in YPD and in SD without uracil and tryptophan; thoseable to grow in YPD, but unable to grow in SD-URA-TRP were selected andtheir tolerance was compared with the original, plasmid containingstrain. As a final confirmation, the plasmid was recovered from thecolonies able to pass the previous controls, transformed into the JM164strain and again selected for those clones giving tolerance. The resultsobtained are summarised in Table 1:

TABLE 1 Number of colonies Number of colonies Drought stress on YPD onSD Number of transformants ≅241000 ≅241000 Transformants isolated 55 40in 1^(st) round Clones with irreproducible 11 2 tolerance Toleranceindependent of the 37 36 plasmid Positive clones confirmed 7 2 byretransformation

The reconfirmed positive clones were sequenced, they encoded threedifferent genes, named Xero1 to Xero3. Xero2 encoded a class-2haemoglobin. A sequence alignment with other plant class-2 haemoglobinsis given in FIG. 1.

Example 4 Xero2 Gives Tolerance to Osmotic Stress, but Also to HighTemperature Stress in Yeast

A dilution series of JM164 pYPGEXero2 and JM164 pYPGE (control) wasplated on YPD medium with 1.7 M sorbitol and tested for osmotictolerance after 2 and 4 days.

The yeast done with Xero2 had a strong sorbitol tolerance phenotype andthe phenotype was very reproducible: at a concentration of 1.7 Msorbitol, control yeast cells did not grow at all, whilst yeast cellsoverexpressing Xero2 did (FIG. 2).

The definition of a strong phenotype is based on drop test experiments.Several dilutions of saturated cultures (1:10, 1:100, 1:1000) were madeand these were grown on selective media (YPD with 1.7M sorbitol).“Strong phenotypes” were those clones that grew well in all thedilutions assayed. With “no strong phenotypes” is meant that the clonedoes not grow in all dilutions. The control cells expressing the emptyplasmid did not grow at all in the selective media.

In the same way, JM164 pYPGEXERO2 and JM164 pYPGE were plated on YPDmedium and incubated at 37° C. for 2 and 3 days. The JM164 pYPGEXERO2clone showed a higher tolerance for elevated temperatures than the wildtype (FIG. 2).

Tolerance to other toxic compounds such as lithium, sodium, hydrogenperoxide, menadione, and tert-Butyl Hydroperoxide (tBOOH) was alsoassayed but without any significative result.

Example 5 Southern Blotting Reveals More than One Isoform in Sugar Beet

In order to confirm the presence of BvXero2 in the sugar beet genome andto estimate the number of genes encoding haemoglobin in this plantspecies, a Southern blot analysis was performed. Genomic DNA wasprepared from leaves of 3-week old sugar beet leaves (Rogers S O andBendich A J, Extraction of total cellular DNA from plants, algae andfungi (Eds) Plant molecular biology manual, Kluwer Academic Publishers,Dordrecht, Netherlands, 1994). 5 μg of DNA was digested with EcoRI,HindIII or BamHI, electrophoresed in 0.8% agarose gel and blotted onto anylon membrane filter (Hybond N+, Amersham Life Science). The membranefilter was hybridised with a ³²P-labelled probe corresponding to the 878bp EcoRI-XhoI digestion fragment of pYPGEhemo, which spans the wholecDNA Hybridisation and washes were carried out under high stringencyconditions (650° C.) (Church G M and Gilbert W., PNAS USA 81: 1991-19951984). The presence of several hybridisation fragments in all lanes,independent of the restriction endonucleases used to digest the genomicDNA, suggested that there are at least two isoforms of BvXero2 in sugarbeet that hybridise with the whole cDNA (FIG. 3). The 878 bp probe mayfurthermore be used to detect and isolate other isoforms of BvXero2.

Example 6 BvXero2 is Induced by NaCl and ABA in Sugar Beet

In order to confirm that BvXero2 participates in the response of sugarbeet plants to salt stress, the accumulation of BvXero2 mRNA in responseto various exposure times to NaCl was analysed using northern blotanalysis. Total RNA was isolated from control, 250 mM Na⁺ or 100 μMABA-treated sugar beet leaves as described by Davis et al. (Basicmethods in Molecular Biology. Elsevier. Amsterdam pp. 143-146 1986). 30μg of total RNA was separated on a 1% agarose gel containing 2.2%formaldehyde and blotted onto a nylon membrane filter (Hybond N,Amersham Life Science). Hybridization was performed using the abovedescribed probe.

The BvXero2 specific probe showed only one band that corresponded to thesize of the BvXero2 cDNA (0.45 kb). The filter was washed twice with4×SSC buffer (0.6 M NaCl, 0.06 M trisodium citrate adjusted to pH=7 withHCl) 0.1% SDS for 5 minutes and twice with 0.4×SSC, 0.1SDS for fiveminutes at 65° C. The same filter was re-hybridised with a 1.9 EcoRIfragment comprising the α₃-tubulin gene of Arabidopsis thaliana (Ludwiget al. Characterization of the α-tubulin gene family of Arabidopsisthaliana PNAS USA 84: 5833-5837 1987). As shown in FIG. 4 the BvXero2mRNA accumulated with time upon NaCl treatment, and reached a maximum at8 hours. The increase was about 10 fold as compared to control plants.This high level was maintained at least until 24 hrs after induction onNaCl. It is interesting to note that the sugar beet cDNA library used tosearch for genes involved in stress tolerance was also obtained fromplants treated for 24 hours with NaCl. An induction of BvXero2 after 3hours of ABA treatment was also observed (FIG. 5). This increase wasobserved even with a huge variation of the background level, which couldbe due to timing, or light induction. The increase was about 2 fold atthree hours, but after six hours BvXERO2 almost disappeared in thecontrol lanes, whilst the ABA treated lanes still showed a significantsignal.

Example 7 Arabidopsis thaliana Transformed with AtHb2 (CDS2591)

Cloning of CDS2591

The nucleic acid CDS2591 was amplified by PCR using an Arabidopsisthaliana seedling cDNA library (Invitrogen, Paisley, UK) as template.After reverse transcription of RNA extracted from seedlings, the cDNAswere cloned into pCMV Sport 6.0. Average insert size of the bank was 1.5kb, and original number of clones was of 1.59×10⁷ cfu. The originaltiter was determined to be 9.6×10⁵ cfu/ml, after a first amplificationit became 6×10¹¹ cfu/ml. After plasmid extraction, 200 ng of templatewas used in a 50 μl PCR mix. Primers prm6122 (SEQ ID NO 14) and prm5458(SEQ ID NO 15), which include the AttB sites for Gateway recombination,were used for PCR amplification. PCR was performed using Hifi Taq DNApolymerase in standard conditions. A PCR fragment of 503 bp wasamplified and purified, also using standard methods. The first step ofthe Gateway procedure, the BP reaction, was then performed, during whichthe PCR fragment recombined in viva with the pDONR plasmid to produce,according to the Gateway terminology, an “entry clone”, p55 (FIG. 6).Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway®technology.

Vector Construction for Transformation with p35 S-CDS2591 Cassette

The entry clone p56 was subsequently used in an LR reaction with p1978,a destination vector used for Arabidopsis transformation. This vectorcontains as functional elements within the T-DNA borders: a plantselectable marker; a GFP expression cassette; and a Gateway cassetteintended for LR in viva recombination with the sequence of interestalready cloned in the entry clone. The p35 S promoter for constitutiveexpression is located upstream of this Gateway cassette.

After the LR recombination step, the resulting expression vector p56(FIG. 7) can be transformed into Agrobacterium strain C58C1RIF PMP90 andsubsequently to Arabidopsis plants.

Transformation of Arabidopsis with p35 S-CDS2591

Sowing and Growing of the Parental Plants

For the parental plants, approximately 12 mg of wild-type Arabidopsisthaliana (ecotype Columbia) seeds was suspended in 27.5 ml of 0.2% agarsolution. The seeds were incubated for 2 to 3 days at a temperature of4° C. and then sown. The plants were germinated under the followingstandard conditions: 22° C. during the day, 18° C. at night, 65-70%relative humidity, 12 hours of photoperiod, sub-irrigation with waterfor 15 min every 2 or 3 days. The seedlings that developed were thentransplanted to pots with a diameter of 5.5 cm, containing a mixture ofsand and peat in a ratio of 1 to 3. The plants were then further grownunder the same standard conditions as mentioned above.

Agrobacterium Growth Conditions and Preparation

Agrobacterium strain C58C1RIF with helper plasmid pMP90 containingvector p56 was inoculated in a 50 ml plastic tube containing 1 ml LB(Luria Broth) without antibiotic. The culture was shaken for 8-9 h at28° C. Hereafter, 10 ml of LB without antibiotic was added to theplastic tube and shaken overnight at 28° C. At an optical density(OD₆₀₀) of approximately 2.0, 40 ml of 10% sucrose and 0.05% Silwet L-77(a mixture of polyalkyleneoxide modified heptamethyltrisiloxane (84%)and allyloxypolyethyleneglycol methyl ether (16%), OSI Specialties Inc)was added to the culture. The Agrobacterium culture so obtained waslabeled CD7659 and used to transform the grown plants.

Flower Dip

When each parental plant had one inflorescence of 7-10 cm in height, theinflorescences were inverted into the Agrobacterium culture and agitatedgently for 2-3 seconds. 2 plants per transformation were used. Theplants were then returned to the normal growing conditions as describedabove.

Seed Collection

5 weeks after the flowers were dipped in the Agrobacterium culture,watering of the plants was stopped. The plants were incubated at 25° C.with a photoperiod of 20 hours. One week later, the seeds were harvestedand placed in a seed drier for one week. The seeds were then cleaned andcollected in 15 ml plastic tubes. The seeds were then stored at 4° C.until further processing.

Growth performance of transgenic Arabidopsis p35 S-CDS2591 plants undersalt stress Seeds harvested from the primarily transformed Arabidopsisplants, here referred to as T0 seeds, were used to evaluate growthperformance under salt stress. Transgenic T1 plants were compared to thesegregant non-transgenic nullizygous plants of the same mother plant,here denominated control plants. The visual marker incorporated into theplants was used to identify transformed and control seeds. To this aimdry seeds were examined under blue light to determine the presence oftransformed seeds. 80 bright fluorescent seeds (expressing thetransgene) and the same amount of non-fluorescent seeds (not expressingthe transgene) seeds were imbibed in 0.2% agar at 4° C. and allowed togerminate in a soil mixture of sand and peat (1:3). At 15 days postimbibition (dpi), a set of 14 individual transgenic and 12 controlplants, all in a similar developmental stage, were selected for furtheranalysis and transplanted to soil on pots of 6.5 cm in diameter. Plantswere grown in greenhouse conditions (22° C. during the day, 18° C. atnight, 60% relative humidity, 20 hour photoperiod, with a sub-irrigationwatering). Salt treatment was applied 3 times over a period of 1 week onseedlings of 21 dpi by watering with 150 mM NaCl, and the plants werethen allowed to recover by watering with tap water. The plants werephotographed weekly and at different angles, using a digital camera overa period of 4 weeks. Images were analysed (the number of pixelscorresponding to plant tissues was recorded for each picture), and usedfor measurement of plant size (plant area and height) using appropriatesoftware.

Results

The salt stress applied affected growth of both transgenic andnon-transgenic control plants. Transgenic plants showed a better growthrecovery from stress as can be derived from FIG. 8. Transgenic plantsshowed a higher growth rate such that four weeks after recovery fromstress treatment the rosette of the transgenic plants was 20% largerthan in control plants (Table 2). However, the strongest effect wasobserved in the development of the inflorescence structures.Non-transgenic plants had a poor inflorescence structure with very fewbranches, while transgenic plants were able to develop theirinflorescence further so that more branches, more flowers, more siliquesand presumably more seeds were produced (FIG. 9). Two parametersreflected the development of the inflorescence, 1) Inflorescence height,which is the distance between 2 horizontal lines drawn at the rosettelevel and the highest point detected for the inflorescence structuresand 2) Inflorescence Area, which is the surface of all plant structuresdetected in the digital images above the rosette level. Values obtainedfor both parameters reflect the better development of inflorescencestructures in transgenic plants and reveals a difference of more than40% in the inflorescence height and of more than 60% in the totalinflorescence area (Table 2).

TABLE 2 Growth performance of transgenic Arabidopsis plants undersalt-stress conditions. Rosette area, Infiorescence height, andInflorescence area are expressed in arbitrary units. The percentagevalues refer to the difference in transgenic plants (TR) with respect tosegregant non- transgenic (NT), taken the values for non transgenicplants as 100. Measurements were done at 45 dpi. The T-test shows thep-value obtained with the student's t-test. Transgenic Non-TransgenicT-test Rosette Area 1787.77 1467.62 0.0120 Rossette Area 121.81 100(TR/NT) In % Inflorescence heigth 110.08 74.67 0.0001 InflorescenceHeigth 147.42 100.00 (TR/NT) In % Inflorescence Area 350.31 217.830.0507 Inflorescence Area 160.81 100 (TR/NT) In %

Example 8 Growth Performance of Rice Plants Transformed with PlantNon-Symbiotic Haemoglobin Coding Sequences

(i) Cloning of Haemoglobin Genes

The isolation of Arabidopsis thaliana heamoglobin gene 2 (AtHB2,CDS2591), and Beta vulgaris haemoglobin gene CDS2767, was described inthe previous examples.

For CDS2767 (Xero2), a plasmid containing the corresponding nucleic acidsequence (earlier described) was used as substrate for the PCR. Specificprimers for each of the haemoglobin genes, detailed in Table 3, wereused in the amplification. In addition to the specific sequences, theforward primers contained the gateway AttB1 site, and the reverseprimers the AttB2 site of the Gateway recombination system.

TABLE 3 List Primers. PrimerNumber Description SEQ ID NO prm05458 attB2CDS2591 7 prm06122 attB1 CDS2591 8 prm06021 attB1 CDS2767 11 prm06022attB2 CDS2767 12

PCR was performed using Hifi Taq DNA polymerase in standard conditions.Specific PCR fragments corresponding to the CDS (Table 4) were isolatedand purified using standard procedures. The first step of the Gatewayprocedure, the BP reaction, was then performed, during which the PCRfragment recombines in vivo with the pDONR201 plasmid to produce,according to the Gateway terminology, an “entry clone”. A list of genesused and their corresponding entry clones is given in Table 4.

TABLE 4 List of haemoglobin genes for cloning into rice. Internal EntryClone reference ORF Origin Species name CDS2591 AtHB2 Arabidopsisthaliana P055 CDS2767 BvHb Beta vulgaris P06289

Each entry clone vector thus contains the corresponding CDS within theAttL1 and AttL2 sites for Gateway® cloning in the pDONR201 backbone.This vector contains also a bacterial kanamycin-resistance cassette anda bacterial origin of replication.

(ii) Vector Construction for Rice Transformation

The entry clones listed in Table 4 were subsequently used in an LRreaction with a destination vector (gateway nomenclature) used for ricetransformation. This vector contains as functional elements within theT-DNA borders: a plant selectable marker, a screenable marker and aGateway cassette intended for LR in vivo recombination with the sequenceof interest already cloned in the entry done. A plant promoter islocated upstream of this Gateway cassette. A description of thedestinations vectors used is given in Table 5.

TABLE 5 Destination vectors. Destination Vector Promoter Descriptionpromoter Expression pattern in rice P00640 PRO0129 Promoter of rice GOS2gene. Constitutive P00831 PRO0218 GenBank AF019212 nucleotides (1-1256)Seed: mainly endosperm P05653 PRO0151 Promoter of rice wsi18 gene. Seed:mainly embryo

After the LR recombination step, the resulting expression vector can betransformed into the Agrobacterium strain LBA4404 and subsequently torice plants since the expression vectors are binary vector forexpression in rice of the different CDS under the control of aparticular promoter. These vectors contain a T-DNA derived from the TiPlasmid, limited by a left border (LB repeat, LB Ti C58) and a rightborder (RB repeat, RB Ti C58)). From the left border to the rightborder, this T-DNA contains: a selectable marker cassette for selectionof transformed plants; a screenable marker cassette for visual screeningof transformed plants; the specific plant promotere-CDS of interest anda zein and rbcS-deltaGA double terminator cassette. These vectors alsocontain an origin of replication from pBR322 for bacterial replicationand a selectable marker (Spe/SmeR) for bacterial selection withspectinomycin and streptomycin. The expression vectors generated and therespective genes of interest are described in Table 6. All constructs (1to 8) are designed for overxpression.

TABLE 6 Plant Transformation Vectors. Entry Destination Construct # doneVector Promotor CDS GOI 1 P06289 P00831 PRO0218 CDS2767 BvHB 2 P06289P00640 PRO0129 CDS2767 BvHB 3 P06289 P05653 PRO0151 CDS2767 BvHB 4P06289 P05653 PRO0151 CDS2591 AtHB2(iii) Transformation of Rice with Plant Transformation Vectors.

The T-DNA of constructs listed in Table 6 were transformed in rice.

Mature dry seeds of Oryza sativa japonica cultivar Nipponbare weredehusked. Sterilization was done by incubating the seeds for one minutein 70% ethanol, followed by 30 minutes in 0.2% HgCl₂ and by 6 washes of15 minutes with sterile distilled water. The sterile seeds were thengerminated on a medium containing 2,4-D (callus induction medium). Aftera 4-week Incubation in the dark, embryogenic, scutellum-derived calliwere excised and propagated on the same medium. Two weeks later, thecalli were multiplied or propagated by subculture on the same medium foranother 2 weeks. 3 days before co-cultivation, embryogenic callus pieceswere sub-cultured on fresh medium to boost cell division activity. TheAgrobacterium strain LBA4404 harbouring the binary vectors (constructs 1to 8, Table 6), was used for co-cultivation. The Agrobacterium strainwas cultured for 3 days at 28° C. on AB medium with the appropriateantibiotics. The bacteria were then collected and suspended in liquidco-cultivation medium at an OD₆₀₀ of about 1. The suspension wastransferred to a petri dish and the calli were immersed in thesuspension during 15 minutes. Next, the callus tissues were blotted dryon a filter paper, transferred to solidified co-cultivation medium andincubated for 3 days in the dark at 25° C. Thereafter, co-cultivatedcallus was grown on 2,4-D-containing medium for 4 weeks in the dark at28° C. in the presence of a selective agent at a suitable concentration.During this period, rapidly growing resistant callus islands developed.Upon transfer of this material to a regeneration medium and incubationin the light, the embryogenic potential was released and shootsdeveloped in the next four to five weeks. Shoots were excised from thecallus and incubated for 2 to 3 weeks on an auxin-containing medium fromwhich they were transferred to soil. Hardened shoots were grown underhigh humidity and short days in a greenhouse. Finally seeds wereharvested three to five months after transplanting. The method yieldedsingle locus transformants at a rate of over 50% (Aldemita and Hodges,Planta 199, 612-617, 1996; Chan et al., Plant Mol. Biol. 22(3), 491-506,1993; Hiei et al., Plant J. 6(2), 271-282, 1994).

(iv) Evaluation of Transgenic Rice Transgenic Plants

Approximately 15 to 20 independent T0 rice transformants are generated.The primary transformants are transferred from tissue culture chambersto a greenhouse for growing and harvest of T1 seed. Generally, 5-10events, of which the T1 progeny segregates 3:1 for presence/absence ofthe transgene, are retained. For each of these events, approximately 10T1 seedlings containing the transgene (hetero- and homo-zygotes), andapproximately 10 T1 seedlings lacking the transgene (nullizygotes), areselected by monitoring expression of the screenable marker.

Vegetative Growth Measurements

The selected T1 plants (approximately 10 with the transgene andapproximately 10 without the transgene) are transferred to a greenhouse.Each plant receives a unique barcode label to link unambiguously thephenotyping data to the corresponding plant. The selected T1 plants aregrown on soil in 10 cm diameter pots under the following environmentalsettings: a photoperiod of 11.5 h, daylight intensity of 30,000 lux ormore, daytime temperature of 28° C. or higher, night time temperature of22° C., relative humidity between 60-70%. Transgenic plants and thecorresponding nullizygotes are grown side-by-side at random positions.From the stage of sowing until the stage of maturity each plant arepassed several times through a digital imaging cabinet and imaged. Ateach time point digital images (2048×1536 pixels, 16 million colors) aretaken of each plant from at least 6 different angles. Also, pictures aretaken from each of the approximately ten selected transgenic plants withthe transgene and also from each of the selected plants not containingthe transgene. One or more of the parameters described below can bederived in an automated way from the all the digital images of all theplants, using image analysis software.

(a) Aboveground Plant Area

Plant above ground area is determined by counting the total number ofpixels from aboveground plant parts discriminated from the background.This value is averaged for the pictures taken on the same time pointfrom the different angles and is converted to a physical surface valueexpressed in square mm by calibration. Experiments show that theaboveground plant area measured this way correlates with the biomass ofplant parts above ground.

(b) Plant Height

Plant height is determined by measuring the distance between thehorizontal lines going through the upper pot edge and the uppermostpixel corresponding to a plant part above ground. This value is averagedfor the pictures taken on the same time point from the different anglesand is converted, by calibration, to a physical distance expressed inmm. Experiments showed that plant height measured this way correlatewith plant height measured manually with a ruler.

(c) Number of Tillers

The number of primary tillers is manually counted at the harvesting ofthe plants. The tillers are cut off at 3 cm above the pot rim. They arethen counted at the cut surface. Tillers that are together in the samesheath are counted as one tiller.

(d) Number of Primary Panicles

The tallest panicle and all the panicles that overlap with the tallestpanicles when aligned vertically are counted manually, and considered asprimary panicles.

(e) Number of Secondary Panicles

The number of panicles that remain on the plant after the harvest of theprimary panicles is counted and considered as secondary panicles.

(f) Growth Curve

The weekly measurements of the plant area are modelled to obtain agrowth curve for each plant, plotted as the value of plant area (in mm²)over the time (in days). From this growth curve the following parameterscan be calculated:

(g) A42

A42 is the plant area at day 42 after sowing as predicted by the growthcurve model.

(h) Tmid

Tmid is the time that a plant needs to grow and reach 50% of the maximumplant area. Tmid is predicted from the growth curve model.

(i) T90

T90 is the time that a plant needs to grow and reach 90% of the maximumplant area. T90 is predicted from the growth curve model.

Seed-Related Parameter Measurements

The mature primary panicles are harvested, bagged, barcode-labelled andthen dried for three days in the oven at 37° C. The panicles are thenthreshed and all the seeds are collected and counted. The filled husksare separated from the empty ones using an air-blowing device. The emptyhusks are discarded and the remaining fraction is counted again. Thefilled husks are weighed on an analytical balance. This procedureresults in the set of seed-related parameters described below.

(a) Total Seed Number Per Plant

Total seed number per plant is measured by counting the number of husksharvested from a plant

(b) Number of Filled Seeds:

Number of filled seeds is determined by counting the number of filledhusks that remaine after the separation step.

(c) Total Seed Yield Per Plant

The total seed yield is measured by weighing all filled husks harvestedfrom a plant.

(d) Harvest Index of Plants

The harvest index in the present invention is defined as the ratiobetween the total seed yield and the above ground area (mm²), multipliedby a factor 10⁶.

(e) Thousand Kernel Weight (TKW) of Plants

This parameter is extrapolated from the number of filled seeds counted,and their total weight.

(f) TotalArea Emergence Prop.

Is the time when the plant reaches 30% of its maximal total area

(g) TotalArea Cycle Time.

Is the time when the plant reaches 90% of its maximal total area

(v) Statistical Analysis: T-Test and F-Test

A two-factor ANOVA (analysis of variants) is used as statistical modelfor the overall evaluation of plant phenotypic characteristics. AnF-test is carried out on all the parameters measured of all the plantsof all the events transformed with the gene of the present invention.The F-test is carried out to check for an effect of the gene over allthe transformation events and to verify for an overall effect of thegene, also named herein “global gene effect”. If the value of the F testshows that the data are significant, than it is concluded that there isa “gene” effect, meaning that not only presence or the position of thegene is causing the differences in phenotype. The threshold forsignificance for a true global gene effect is set at 5% probabilitylevel for the F test.

To check for an effect of the genes within an event, i.e., for aline-specific effect, a t-test is performed within each event using datasets from the transgenic plants and the corresponding null plants. “Nullplants” or “Null segregants” are the plants treated in the same way asthe transgenic plant, but from which the transgene has segregated. Nullplants can also be described as the homozygous negative transformants.The threshold for significance for the t-test is set at 10% probabilitylevel. Within one population of 5 transformation events, some events canbe under or above this t-test threshold. This is based on the hypothesisthat a gene might only have an effect in certain positions in thegenome, and that the occurrence of this position-dependent effect is notuncommon. This kind of gene effect is also named herein a “line effectof the gene”.

The p value is obtained by comparing the t value to the t distributionor alternatively, by comparing the F value to the F distribution. The pvalue then stands for the probability that the null hypothesis (nullhypothesis being “there is no effect of the transgene”) is correct.

(vi) Specific Growth Conditions.

Growth of the plants takes place in the greenhouse as described earlier,either under optimal or sub-optimal conditions. Under optimal conditionsthe plants are watered regularly with a nutrient solution containingN:P:K 20:20:20 at a final conductivity of EC=1.1 mS.

Two type of sub-optimal conditions are used:

1—Salt Stress.

The nutrient solution is supplemented with NaCl to 15 mM. The salinesolution is applied to the plants from 3 weeks after sowing till thetime of harvest.

2—Drought Stress.

Plants are grown and watered with a frequency optimal for growth suchthat no signs of excess or deficit of watered are visible, till headingstage. When about 50% of the control non-transgenic plants for aparticular construct reach the end of tillering and panicles start toform at heading stage (end of stage V9 to R0 stage as defined by Counceet al. 2000), all plants (transgenic and non-transgenic) belonging thatparticular construct are watered till the pots contain 60% RWC (relativewater content), then water is withheld till the RWC drops to 20% (mostcontrol plants show a rolling index of 4). Rewatering to normal optimalfrequency is then resumed

Transgenic plants showing improved values from an agronomical point ofview, for a biomass or seed parameter measured in the greenhouse, areselected for further testing in field conditions.

1. Method for producing plants with at least one altered plantcharacteristic selected from increased yield, increased biomass, alteredarchitecture and altered cell division of the plant compared to acontrol plant, said method comprising increasing expression in plants ofa nucleic acid sequence encoding plant class-2 non-symbiotic haemoglobinby transforming said plants with a nucleic acid sequence encoding plantclass-2 non-symbiotic haemoglobin to produce transformed plants, saidnucleic acid sequence being SEQ ID NO:3 or a nucleic acid sequenceencoding the amino acid sequence of SEQ ID NO:4, wherein the plantcharacteristic is selected from one or more of increased yield,increased biomass, altered architecture or altered cell division, andselecting from the transformed plants those transformed plants whichhave at least one of said characteristics.
 2. Method of claim 1, whereinsaid increased yield comprises increased seed yield.